Complex formation method and separation method

ABSTRACT

An object of the present invention is to provide a method for forming a complex between an analyte or an analog thereof, and a substance formable the complex with said analyte or said analog thereof (the complex forming substance), in a short time and in high reaction efficiency, and a method for separating a complex formed, and a complex forming substance not involved in formation of said complex or an analog not involved in formation of said complex rapidly, simply and in high accuracy, along with a method for measuring an analyte in a sample in high sensitivity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application of PCT/US2006/033043,filed Aug. 24, 2006, which claims priority from U.S. ProvisionalApplication No. 60/713,346, filed Sep. 2, 2005.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to a method for forming a complex betweenan analyte or an analogue thereof in a sample, and a substance formablea complex with said analyte or analogue thereof (hereinafter,abbreviated as a complex forming substance or CFS), a method forseparating a complex formed, and a CFS or an analogue not involved information of said complex, along with a method for measuring an analytein a sample, based on the amount of a complex separated, or the amountof a CFS or an analogue not involved in formation of a complex.

2. Background Art

Analysis of an analyte in a sample usually requires mixing a pluralityof solutions such as a sample and various reagent solutions (forexample, a reagent solution including an antibody to an analyte, areagent solution containing a labeling substance, and the like) etc.,and subjecting an analyte in a sample and a reactant in a reagentsolution (an antibody to an analyte or a labeling substance, and thelike) to a reaction.

In Micro Total Analysis System (μ-TAS) using micro fluidics device,wherein, technology thereof has recently been developing and variousresearches thereon have been made, a method for mixing these pluralityof solutions in advance and subjecting to a reaction outside a capillary(channel), then introducing the mixed solution into a capillary(channel), or a method for simultaneously introducing a plurality ofsolutions into a mixing capillary (channel) to carry out mixing andreaction (Patent Literature 1) has been known.

However, in the former method, a sample or a reagent solution in theamount of micro litter (μl) order is required for mixing in advance,which loses merit of μ-TAS, namely possibility of micro analysis of asample or a reagent solution in the amount of from nano litter (nl) topico litter (pl) order. In addition, in the latter method, laminar flowgenerating in a capillary in introduction makes mixing of a plurality ofsolutions difficult, resulting in requirement to depend on moleculardiffusion, which poses such a problem as, in the case of mixing aplurality of solutions with different molecular weight or viscosity, thevariation of complete mixing time of these solutions due to variation ofdiffusion coefficient, or variation in viscosity ratio of solutions tobe mixed varies volume ratio of a plurality of solutions to beintroduced in a channel, which results in variation of mixing ratiodepending on kinds of solutions to be mixed, which poses a problem ofmaking mixing in constant mixing ratio impossible.

In addition, as a method other than the above, there are methodsdisclosed in Bao, J. M, Regnier, F. E, J. Chromatogr. 1992, 608, 217-224(Non-Patent Literature 1) or JP-A-10-512371 (Patent Literature 2).

In these methods, in a capillary for analysis, a solution including amolecule with higher electrophoretic mobility is arranged at thebackward of a solution containing a molecule with lower electrophoreticmobility, so that a molecule with higher electrophoretic mobilityovertakes a molecule with lower electrophoretic mobility, by applicationof electric field, by which a reaction between these moleculesthemselves is carried out. These methods make possible molecular mixinguniformly and in a short time compared with conventional mixing methodsdepending on molecular diffusion.

However, these methods are not satisfactory in reaction efficiency, andcountermeasures to ensure sufficient reaction efficiency is required todetect an analyte in a sample in high sensitivity, for example, toincrease concentration of molecules to be fed to a reaction, or tolonger reaction time by delaying molecular movement rate byelectrophoresis.

Patent Literature 1: JP-A-2005-31070

Patent Literature 2: JP-A-10-512371

Non-Patent Literature 1: Bao, J. M, Regnier, F. E, J. Chromatogr. 1992,608, 217-224

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The present invention relates to a method for forming a complex betweensaid analyte or analogue thereof, and said CFS in a short time and inhigh reaction efficiency, a method for separating a complex formed, anda CFS or an analogue not involved in formation of said complex quickly,easily and with a high degree of accuracy, along with a method formeasuring an analyte in a sample in high sensitivity.

Means for Solving Problems

The present invention is composed of the following framework:

1. A Method for Forming a Complex Comprising the Following Steps:

-   -   (1) a step of arranging (a) a solution containing an analyte or        an analogue thereof and (b) a solution containing not less than        one kind of a CFS, in a capillary, so that by applying a voltage        to said capillary the complex between said analyte or said        analogue thereof and the CFS are formed without mixing these        solutions in advance; and    -   (2) a step of contacting said analyte or said analogue thereof        with the CFS while concentrating said analyte or said analogue        thereof and/or at least one kind of the CFSs by applying a        voltage to said capillary before uniformly mixing these        solutions to form the complex between said analyte or said        analogue thereof and the CFS.

2. A Method for Separating a Complex Comprising the Following Steps:

-   -   (1) a step of arranging (a) a solution containing an analyte or        an analogue thereof and (b) a solution containing not less than        one kind of a CFS, in a capillary, so that by applying a voltage        to said capillary the complex between said analyte or said        analogue thereof and the CFS are formed without mixing these        solutions in advance;    -   (2) a step of contacting said analyte or said analogue thereof        with the CFS while concentrating said analyte or said analogue        thereof and/or at least one kind of the CFSs by applying a        voltage to said capillary before uniformly mixing these        solutions to form the complex between said analyte or said        analogue thereof and the CFS; and    -   (3) a step of separating said complex, and the CFS not involved        in the formation of said complex or the analogue not involved in        the formation of said complex by further electrical movement.

3. A Method for Measuring an Analyte Comprising the Following Steps:

-   -   (1) a step of arranging (a) a solution containing an analyte or        an analogue thereof and (b) a solution containing not less than        one kind of a CFS, in a capillary, so that by applying a voltage        to said capillary the complex between said analyte or said        analogue thereof and the CFS are formed without mixing these        solutions in advance;    -   (2) a step of contacting said analyte or said analogue thereof        with the CFS while concentrating said analyte or said analogue        thereof and/or at least one kind of the CFSs by applying a        voltage to said capillary before uniformly mixing these        solutions to form the complex between said analyte or said        analogue thereof and the CFS;    -   (3) a step of separating said complex, and the CFS not involved        in the formation of said complex or the analogue not involved in        the formation of said complex by further electrical movement;        and    -   (4) a step of measuring the amount of thus separated complex, or        the amount of the CFS or the analogue not involved in the        formation of said complex to determine the amount of said        analyte based on the result.

Namely, the present inventors found that by arranging a solutioncontaining an analyte or an analogue thereof, and a solution containinga CFS, in a channel, without mixing these solutions in advance, to forma complex between said analyte and analogue, and CFS while concentratingsaid analyte or analogue thereof and/or CFS electrophoretically byapplying a voltage onto this capillary, by utilization of difference inelectrophoretic mobility, said complex can be formed in a short time andin high reaction efficiency by using only ultra small amount of a sampleand said solution from nano litter (nl) to pico litter (pl) order, andwithout taking consideration on variation in mixing ratio caused bydifference in viscosity of a sample and said solution, and have thuscompleted the present invention.

Effect of the Invention

In accordance with a method of the present invention, a reaction betweenan analyte or an analogue thereof in a solution and a CFS in a solution,can be carried out in a short time and in high reaction efficiency. As aresult, separation of a complex with a CFS, and a CFS or an analogue notinvolved in formation of a complex becomes possible rapidly, simply andin high accuracy, and furthermore, high sensitivity measurement of ananalyte in a sample becomes possible, based on the amount of separatedcomplex or the amount of a CFS or analogue not involved in formation ofa complex.

BEST MODES FOR CARRYING OUT THE INVENTION

1. A Method for Forming a Complex of the Present Invention

A method of the present invention is characterized in that (a) asolution containing an analyte or an analogue thereof, and a solutioncontaining a CFS are introduce and arranged into each separate zone in acapillary, without forming a complex between them by mixing thesesolutions in advance outside a capillary, and subsequently (b) a complexbetween said analyte or analogue thereof and CFS is formed whileconcentrating said analyte or analogue thereof and/or CFSelectrophoretically to make contact these by applying a voltage ontosaid channel, before uniformly mixing these solutions in a capillary

A method for forming a complex of the present invention specificallycomprises the following step (1) and step (2):

(1) a step (a step of introduction) of arranging (a) a solutioncontaining an analyte or an analogue thereof and (b) a solutioncontaining not less than one kind of a CFS with said analyte or saidanalogue thereof, in a capillary, so that by applying a voltage to saidcapillary the complex between said analyte or said analogue thereof andthe CFS are formed without mixing these solutions in advance; and(2) a step (a step of concentrating reaction) of contacting said analyteor said analogue thereof with the CFS while concentrating said analyteor said analogue thereof and/or at least one kind of the CFSs byapplying a voltage to said capillary before uniformly mixing thesesolutions to form the complex between said analyte or said analoguethereof and the CFS.1-1. A Step of Introduction [a Step (1)]

A step (1) of the present invention is a step of introducing andarranging a solution containing an analyte or an analogue thereof, andsolutions containing not less than one kind of CFSs into a capillary, sothat a complex between an analyte or an analogue thereof and a CFS isformed, without mixing these solutions in advance outside a capillary,and by applying a voltage onto a capillary, namely by carrying out astep (2) of the present invention as described later.

Here, “so that a complex between an analyte or an analogue thereof and aCFS is formed, by applying a voltage onto a capillary” means to form acomplex between an analyte or an analogue thereof and a CFS bycontacting the analyte or analogue thereof with the CFS, not by (notdepending on) molecular diffusion and by utilization of the phenomenonthat when a solution containing a substance with higher electrophoreticmobility (faster electrophoretic speed) is arranged at upstream of asolution containing a substance with lower electrophoretic mobility(slow electrophoretic speed) and electrophoresis is carried out, asubstance with higher electrophoretic mobility (faster electrophoreticspeed) in a solution overtakes a substance with lower electrophoreticmobility (slow electrophoretic speed).

Namely, the present invention aims at forming a complex between (1) ananalyte or an analogue thereof, or a complex between an analyte or ananalogue thereof, and a certain CFS, and (2) at least one kind of a CFS(note: a CFS different from one described above) in a capillary byapplying a voltage onto a capillary. In other words, the presentinvention includes not only the case that a complex between an analyteor an analogue thereof, and all of CFSs is formed only in a capillary,but also such a case is also included, for example, that when 2 or morekinds of CFSs are used, a complex (an intermediate complex) between ananalyte or an analogue thereof, and a part of CFSs among 2 or more kindsof CFSs is formed in advance outside a capillary, or in a capillarywithout application of a voltage, and subsequently said intermediatecomplex and residual not less than one kind of CFSs are made contactedin a capillary by applying a voltage onto a capillary, to form a complexbetween the intermediate complex formed in advance, and the residual notless than one kind of CFSs.

For example, when 2 kinds of CFSs are used, such a case is naturallyincluded that (1) a complex (an intermediate complex) between an analyteor an analogue thereof and one kind of a CFS, and a complex between saidintermediate complex and a residual one kind of a CFS is formed in acapillary by applying a voltage onto a capillary, and such a case isalso included that (2) an intermediate complex between an analyte or ananalogue thereof, and a kind of a CFS is formed in advance outside acapillary, or in a capillary without application of a voltage, andsubsequently said intermediate complex and a residual one kind of a CFSis formed in a capillary by applying a voltage onto a capillary. Inaddition, for example, when 3 kinds of CFSs are used, the case isnaturally included that (1) a complex (an intermediate complex 1)between an analyte or an analogue thereof and one kind of a CFS (aCFS-1), a complex (an intermediate complex 2) between said intermediatecomplex 1 and the residual one kind of a CFS (a CFS-2), and a complexbetween the intermediate complex 2 and residual one kind of a CFS (aCFS-3) are formed in a capillary by applying a voltage onto a capillary,and such cases are also included that (2) an intermediate complex 1between an analyte or an analogue thereof, and a CFS-1 is formed inadvance outside a capillary, or in a capillary without application of avoltage, and then an intermediate complex 2 between said intermediatecomplex 1 and a CFS-2, along with a complex between said intermediatecomplex 2 and a CFS-3 are formed in a capillary by applying a voltageonto a capillary; or (3) an intermediate complex 1 between an analyte oran analogue thereof and a CFS-1, and an intermediate complex 2 betweensaid intermediate complex 1 and a CFS-2 are formed in advance outside acapillary, or in a capillary without application of a voltage, and then,a complex between said intermediate complex 2 and a CFS-3 is formed in acapillary by applying a voltage onto a capillary. (In this connection,the cases when 4 or more kinds of CFSs are used are considered by thesame way of thinking.)

Therefore, in the present invention, “without mixing solutions inadvance” means no mixing of a solution containing an analyte or ananalogue thereof, and a solution containing at least one kind of a CFSin advance, but does not necessarily mean exclusion of any mixing of asolution containing an analyte or an analogue thereof, and a solutioncontaining at least one kind of a CFS in advance (in other words, itdoes not mean never to do to mix some of a sample including an analyteand solutions containing all of CFSs, along with if necessary, asolution containing analogues).

In the present invention, a direction toward which a complex between ananalyte or an analogue thereof, and not less than one kind of CFSs,finally formed when voltage is applied, moves is defined as “downstream”side, and the opposite direction is defined as “upstream” side (the samehereinafter).

In addition, in the present invention, “electrophoretic speed (of ananalyte or an analogue thereof, a CFS, and the like are subjected toelectrophoresis) is “slow” or “electrophoretic mobility (of an analyteor an analogue thereof, a CFS, and the like are subjected toelectrophoresis) is low” means not only the case when electrophoreticspeed is slow (electrophoretic mobility is low) than those of at leastnot less than one kind other substances, but also means movement in adirection opposite to the direction of at least not less than one kindother substances.

Arrangement order of a solution containing an analyte or an analoguethereof, and solutions including not less than one kind of CFSs is notespecially limited, as long as it is an order formable a complex betweenan analyte or an analogue thereof, and a CFS by applying a voltage ontoa capillary.

In Tables 1-1 to 1-5, relations between arrangement order of a solutioncontaining an analyte or an analogue thereof and a solution including aCFS, and electrophoretic mobility (electrophoretic speed) of an analyteor an analogue thereof, and a CFS are shown, however, the presentinvention is by no means limited thereto.

In this connection, in Tables 1-1 to 1-5, the cases when from one kindto 3 kinds of CFSs are used are shown, however, the same way of thinkingas in Tables 1-1 to 1-5 is applied in suitable arrangement, also in thecase when 4 or more kinds of CFSs are used.

In Tables 1-1 to 1-5, Ana represents an analyte or an analogue thereof,CFS-1 represents a CFS-1, CFS-2 represents a CFS-2, and CFS-3 representsa CFS-3. In addition, in Tables 1-1 to 1-5, arrangement order A in acapillary is a zone of a solution arranged at the most upstream side,among a solution containing an analyte or an analogue thereof, andsolutions including not less than one kind of CFSs, Bis a zone of asolution arranged at the downstream side of zone A, C is a zone of asolution arranged at the downstream side of zone B, and D is a zone of asolution arranged at the downstream side of zone C. In this connection,the arrangement orders (A to D) in said capillary is only the ordersamong a solution containing an analyte or an analogue thereof, andsolutions including not less than one kind of CFSs, and such arrangementmay naturally be allowed that a solution, and the like, other than saidsolutions is arranged at further downstream side of said solutionarranged at the most downstream side among these, or at further upstreamside of said solution arranged at the most upstream side.

TABLE 1-1 Arrangement order in capillary (Upstream→Downstream)Relationship of Electorophoretic Pattern A B C D Mobility between Anaand CFS 1 Solution Solution — — Ana > CFS-1 containing containing AnaCFS-1 2 Solution Solution — — Ana < CFS-1 containing containing CFS-1Ana 3 Solution Solution Solution — (1) Ana > CFS-1; containingcontaining containing (Ana/CFS-1)complex > CFS-2 Ana CFS-1 CFS-2However, (Ana/CFS-1)complex is formed prior to (Ana/CFS-2)complex. 4 (2)CFS-1 > Ana > CFS-2; (Ana/CFS-2)complex > CFS-1 However,(Ana/CFS-2)complex is formed prior to (Ana/CFS-1)complex. 5 SolutionSolution Solution — (1) Ana < CFS-1; containing containing containing(Ana/CFS-1)complex > CFS-2 CFS-1 Ana CFS-2 However, (Ana/CFS-1)complexis formed prior to (Ana/CFS-2)complex. 6 (2) Ana > CFS-2;(Ana/CFS-2)complex < CFS-1 However, (Ana/CFS-2)complex is formed priorto (Ana/CFS-1)complex. 7 Solution Solution Solution — (1) CFS-2 < CFS-1;containing containing containing Ana < CFS-1(Ana/CFS-1)complex < CFS-1CFS-2 Ana CFS-2 However, (Ana/CFS-1)complex is formed prior to(Ana/CFS-2)complex. 8 (2) Ana < CFS-2; (Ana/CFS-2)complex < CFS-1However, (Ana/CFS-2)complex is formed prior to (Ana/CFS-1)complex.

TABLE 1-2 9 Solution Solution Solution Solution (1) Ana > CFS-1;containing containing containing containing (Ana/CFS-1)complex > CFS-2;Ana CFS-1 CFS-2 CFS-3 (Ana/CFS-1/CFS-2) complex > CFS-3 However,(Ana/CFS-1)complex is formed prior to (Ana/CFS-2)complex and(Ana/CFS-3)complex, (Ana/CFS-1/CFS-2)complex is formed priorto(Ana/CFS-1/CFS-3)complex. 10 (2) Ana > CFS-1; CFS-2 >(Ana/CFS-1)complex > CFS-3; (Ana/CFS-1/CFS-3)complex > CFS-2 However,(Ana/CFS-1)complex is formed prior to (Ana/CFS-2)complex and(Ana/CFS-3)complex, (Ana/CFS-1/CFS-3)complex is formed prior to(Ana/CFS-1/CFS-2)complex. 11 (3) CFS-1 > Ana > CFS-2;(Ana/CFS-2)complex > CFS-1; (Ana/CFS-2/CFS-1)complex > CFS-3 However,(Ana/CFS-2)complex is formed prior to (Ana/CFS-1)complex and(Ana/CFS-3)complex, (Ana/CFS-2/CFS-1)complex is formed prior to(Ana/CFS-2/CFS-3)complex. 12 (4)CFS-1 > Ana > CFS-2; CFS-1 >(Ana/CFS-2)complex > CFS-3; (Ana/CFS-2/CFS-3)complex > CFS-1 However,(Ana/CFS-2)complex is formed prior to (Ana/CFS-1)complex and(Ana/CFS-3)complex, (Ana/CFS-2/CFS-3)complex is formed priorto(Ana/CFS-2/CFS-1)complex. 13 (5) CFS-1, CFS-2 > Ana > CFS-3;(Ana/CFS-3)complex > CFS-1; (Ana/CFS-3/CFS-1)complex > CFS-2 However,(Ana/CFS-3)complex is formed prior to (Ana/CFS-1)complex and(Ana/CFS-2)complex, (Ana/CFS-3/CFS-1)complex is formed prior to(Ana/CFS-3/CFS-2)complex. 14 (6) CFS-1, CFS-2 > Ana > CFS-3; CFS-1 >(Ana/CFS-3)complex > CFS-2; (Ana/CFS-3/CFS-2)complex > CFS-1 However,(Ana/CFS-3)complex is formed prior to (Ana/CFS-1)complex and(Ana/CFS-2)complex, (Ana/CFS-3/CFS-2)) complex is formed prior to(Ana/CFS-3/CFS-1)complex.

TABLE 1-3 15 Solution Solution Solution Solution (1) Ana < CFS-1;containing containing g containing containing (Ana/CFS-1)complex >CFS-2; CFS-1 Ana CFS-2 CFS-3 (Ana/CFS-1/CFS-2)complex > CFS-3 However,(Ana/CFS-1)complex is formed prior to (Ana/CFS-2)complex and(Ana/CFS-3)complex, (Ana/CFS-1/CFS-2)complex is formed prior to(Ana/CFS-1/CFS-3)complex. 16 (2) Ana < CFS-1; CFS-2 >(Ana/CFS-1)complex > CFS-3; (Ana/CFS-1/CFS-2)complex > CFS-3 However,(Ana/CFS-1)complex is formed prior to (Ana/CFS-2)complex and(Ana/CFS-3)complex, (Ana/CFS-1/CFS-3)complex is formed priorto(Ana/CFS-1/CFS-2)complex. 17 (3) Ana > CFS-2; (Ana/CFS-2)complex <CFS-1; (Ana/CFS-2/CFS-1)complex > CFS-3 However, (Ana/CFS-2)complex isformed prior to (Ana/CFS-1)complex and (Ana/CFS-3)complex,(Ana/CFS-2/CFS-1)complex is formed prior to (Ana/CFS-2/CFS-3)complex. 18(4) Ana > CFS-2; (Ana/CFS-2)complex > CFS-3; (Ana/CFS-2/CFS-3)complex <CFS-1 However, (Ana/CFS-2)complex is formed prior to (Ana/CFS-1)complexand (Ana/CFS-3)complex, (Ana/CFS-2/CFS-3)complex is formed prior to(Ana/CFS-2/CFS-1)complex. 19 (5) CFS-2 > Ana > CFS-3; (Ana/CFS-3)complex< CFS-1; (Ana/CFS-3/CFS-1)complex > CFS-2 However, (Ana/CFS-3)complex isformed prior to (Ana/CFS-1)complex and (Ana/CFS-2)complex,(Ana/CFS-3/CFS-1)complex is formed prior to (Ana/CFS-3/CFS-2)complex. 20(6) CFS-2 > Ana > CFS-3; (Ana/CFS-3)complex > CFS-2;(Ana/CFS-3/CFS-2)complex < CFS-1 However, (Ana/CFS-3)complex is formedprior to (Ana/CFS-1)complex and (Ana/CFS-2)complex,(Ana/CFS-3/CFS-2)complex is formed prior to (Ana/CFS-3/CFS-1)complex.

TABLE 1-4 21 Solution Solution Solution Solution (1) CFS-2 < CFS-1;containing containing containing g containing Ana < CFS-1; CFS-1 CFS-2Ana CFS-3 (Ana/CFS-1)complex < CFS-2; (Ana/CFS-1/CFS-2)complex > CFS-3However, (Ana/CFS-1)complex is formed prior to (Ana/CFS-2)complex and(Ana/CFS-3)complex, (Ana/CFS-1/CFS-2)complex is formed priorto(Ana/CFS-1/CFS-3)complex. 22 (2) CFS-2 < CFS-1; Ana < CFS-1;(Ana/CFS-1)complex > CFS-3; (Ana/CFS-1/CFS-3)complex < CFS-2 However,(Ana/CFS-1)complex is formed prior to (Ana/CFS-2)complex and(Ana/CFS-3)complex, (Ana/CFS-1/CFS-3)complex is formed prior to(Ana/CFS-1/CFS-2)complex. 23 (3) Ana < CFS-2; (Ana/CFS-2)complex <CFS-1; (Ana/CFS-2/CFS-1)complex > CFS-3 However, (Ana/CFS-2)complex isformed prior to (Ana/CFS-1)complex and (Ana/CFS-3)complex,(Ana/CFS-2/CFS-1)complex is formed prior to (Ana/CFS-2/CFS-3)complex. 24(4) Ana < CFS-2: (Ana/CFS-2)complex > CFS-3; (Ana/CFS-2/CFS-3)complex >CFS-1 However, (Ana/CFS-2)complex is formed prior to (Ana/CFS-1)complexand (Ana/CFS-3)complex, (Ana/CFS-2/CFS-3)complex is formed prior to(Ana/CFS-2/CFS-1) complex. 25 (5) Ana > CFS-3; CFS-1 > CFS-2;(Ana/CFS-3)complex < CFS-1; (Ana/CFS-3/CFS-1)complex < CFS-2 However,(Ana/CFS-3)complex is formed prior to (Ana/CFS-1)complex and(Ana/CFS-2)complex, (Ana/CFS-3/CFS-1)complex is formed priorto(Ana/CFS-3/CFS-2)complex. 26 (6) Ana > CFS-3; (Ana/CFS-3)complex <CFS-2; (Ana/CFS-3/CFS-2)complex < CFS-1 However, (Ana/CFS-3)complex isformed prior to (Ana/CFS-1)complex and (Ana/CFS-2)complex,(Ana/CFS-3/CFS-2)complex is formed prior to (Ana/CFS-3/CFS-1)complex.

TABLE 1-5 27 Solution Solution Solution Solution (1) CFS-1 > CFS-2,CFS-3, Ana; containing containing containing containing CFS-2 > CFS-3;CFS-1 CFS-2 CFS-3 Ana (Ana/CFS-1)complex < CFS-2;(Ana/CFS-1/CFS-2)complex < CFS-3 However, (Ana/CFS-1)complex is formedprior to (Ana/CFS-2)complex and (Ana/CFS-3)complex,(Ana/CFS-1/CFS-2)complex is formed prior to (Ana/CFS-1/CFS-3)complex. 28(2) CFS-1 > CFS-2, CFS-3, Ana; (Ana/CFS-1)complex < CFS-3;(Ana/CFS-1/CFS-3)complex < CFS-2 However, (Ana/CFS-1)complex is formedprior to (Ana/CFS-2)complex and (Ana/CFS-3)complex,(Ana/CFS-1/CFS-3)complex is formed prior to (Ana/CFS-1/CFS-2)complex. 29(3) CFS-2 > CFS-1, CFS-3, Ana; CFS-1 > CFS-3; (Ana/CFS-2)complex <CFS-1; (Ana/CFS-2/CFS-1)complex < CFS-3 However, (Ana/CFS-2)complex isformed prior to (Ana/CFS-1)complex and (Ana/CFS-3)complex,(Ana/CFS-2/CFS-1)complex is formed prior to (Ana/CFS-2/CFS-3)complex. 30(4) CFS-2 > CFS-1, CFS-3, Ana; (Ana/CFS-2)complex < CFS-1;(Ana/CFS-2/CFS-3)complex < CFS-1 However, (Ana/CFS-2)complex is formedprior to (Ana/CFS-1)complex and (Ana/CFS-3)complex,(Ana/CFS-2/CFS-3)complex is formed prior to (Ana/CFS-2/CFS-1)complex. 31(5) CFS-3 > Ana; CFS-1 > CFS-3; (Ana/CFS-3)complex < CFS-1;(Ana/CFS-3/CFS-1)complex < CFS-2 However, (Ana/CFS-3)complex is formedprior to (Ana/CFS-1)complex and (Ana/CFS-2)complex,(Ana/CFS-3/CFS-1)complex is formed prior to (Ana/CFS-3/CFS-2)complex. 32(6) CFS-3 > Ana; (Ana/CFS-3)complex < CFS-2; (Ana/CFS-3/CFS-2)complex <CFS-1 However, (Ana/CFS-3)complex is formed prior to (Ana/CFS-1)complexand (Ana/CFS-2)complex, (Ana/CFS-3/CFS-2)complex is formed prior to(Ana/CFS-3/CFS-1)complex.

In Tables 1-1 to 1-5, “formed prior to” means that the former complex isformed substantially before the latter complex is formed, but does notexclude when a part of the former complex and the latter complex areformed simultaneously or in reversed order, for example, in thefollowing case.

(1) The case that by adjacently placing each zone (zone of a solutioncontaining an analyte or an analogue thereof, zone of solutionscontaining CFSs) and by molecular diffusion generated at the vicinity ofliquid-liquid interface between each zone among these, a part of theformer complex and the latter complex are formed simultaneously or inreversed order.

(2) The case that from the relation of electrophoretic speed of ananalyte or an analogue thereof and not less than one kind of CFSs, apart of the former complex and the latter complex are formedsimultaneously or in reversed order.

(3) The case that because at least one kind of a CFS among an analyte oran analogue thereof and not less than one kind of CFSs move in adirection opposite from those of other substances, a part of the formercomplex and the latter complex are formed simultaneously or in reversedorder.

In such cases, a solution containing an analyte or an analogue thereof,and solutions containing CFSs may suitably be arranged by the same wayof thinking as in Tables 1-1 to 1-5.

In addition, for the case when 2 or more kinds of CFSs are used, amongthe cases in Tables 1-1 to 1-5, such a case is shown that all of CFSs (aCFS-1 to -3) bind to an analyte or an analogue thereof, namely all ofthe binding sites of 2 or more kinds of CFSs are present only at ananalyte or an analogue thereof [binding form (1): a sandwich complexwith Ana sandwiched by CFS-1 and CFS-2]. The following other cases maybe understood as follows: A case, for example, a CFS-1 in 2 kinds ofCFSs (a CFS-1 and -2) binds to an analyte or an analogue thereof, and aCFS-2 binds to new sites generated by formation of a complex between ananalyte or an analogue thereof, and a CFS-1, in other words, bindingsites of at least one kind of a CFS (for example, a CFS A) among 2 ormore kinds of CFSs are present only on an analyte or an analoguethereof, and binding sites of other at least one kind of a CFS (forexample, a CFS B) are present at new sites generated by formation of acomplex between an analyte or an analogue thereof, and a CFS A, [bindingform (2)]; or a case, for example, a CFS-1 in 2 kinds of CFSs (a CFS-1and -2) binds to an analyte or an analogue thereof, and a CFS-2 binds toa CFS-1 bound with an analyte or an analogue thereof, in other words,binding sites of at least one kind of a CFS (for example, a CFS A) among2 or more kinds of CFSs are present only on an analyte or an analoguethereof, and binding sites of other at least one kind of a CFS (forexample, a CFS B) are present only on a CFS A, [binding form (3)]; arethe cases when a sandwich complex of CFS-1, sandwiched by Ana and CFS-2,is formed in the binding form (1) [an analyte or an analogue thereof inthe case of using 2 kinds of CFSs in Tables 1-1 to 1-5 is alternativelyread as a CFS-1, and a CFS-1 in Tables 1-1 to 1-5 is alternatively readas an analyte or an analogue thereof]. In addition, in such cases as 3kinds or more CFSs are used in the above-described binding forms (2) and(3), or such cases as the above-described binding forms (1) to (3) aresuitably combined, a solution containing an analyte or an analoguethereof, and solutions containing CFSs may suitably be arranged by thesame way of thinking as in Tables 1-1 to 1-5 and the above.

In this connection, in carrying out the present invention in acompetitive method using an analogue of an analyte, any of the followingarrangements may also be adopted; An analyte in a sample (namely, asample including an analyte) and an analogue [for example, an analoguelabeled by a labeling substance (a labeled analogue), and an analoguebound with a reaction improvement substance (a reaction improvementanalogue)], are simultaneously present in the same solution andintroduced and arranged into a capillary as one solution zone (namely, azone of a solution containing a sample including an analyte and ananalogue); a sample including an analyte, and a solution containing ananalogue (for example, a labeling analogue or a reaction improvementanalogue) are introduced and arranged into a capillary as each separatezone (solution); or a solution containing an analyte in a sample(namely, a sample including an analyte) and not less than one kind ofCFSs, and a solution including an analogue (for example, a labeledanalogue or a reaction improvement analogue) are introduced and arrangedinto a capillary as each separate zone (solution).

In this connection, in the above-described competitive method, toarrange, in a capillary, (1) a solution including a sample having ananalyte and a labeled analogue (a solution containing an analyte and ananalogue), along with solutions containing not less than one kind ofCFSs (a CFS, a reaction improvement CFS and combinations thereof); or(2) a sample including an analyte (a solution containing an analyte oran analogue thereof), along with a solution containing a labeledanalogue and solutions including not less than one kind of CFSs (a CFS,a reaction improvement CFS and combinations thereof); or (3) a solutionincluding a sample having an analyte and not less than one kind of CFSs(a CFS, a reaction improvement CFS and combinations thereof), along witha solution containing a labeled analogue; so that a complex A betweensaid analyte and CFS, and a complex B between said labeled analogue andCFS are formed by applying a voltage onto a channel, arrangement orderof these solutions may be determined, in accordance with arrangementorder of a solution including an analyte or an analogue thereof, andsolutions containing not less than one kind of CFSs, as explained above,by consideration on electrophoretic mobility of an analyte, a labeledanalogue and a CFS. In addition, to arrange, in a capillary, (1) asolution including a sample having an analyte and a reaction improvementanalogue (a solution containing an analyte and an analogue), along witha solution containing not less than one kind of a CFS having propertycapable of forming a complex with an analyte or an analogue thereof andlabeled with a labeling substance (hereinafter, abbreviated as a labeledbinding substance or a labeled CFS); or (2) a sample including ananalyte (a solution containing an analyte or an analogue), along with asolution including a reaction improvement analogue and a solutioncontaining not less than one kind of labeled CFSs; or (3) a solutionincluding a sample having an analyte and not less than one kind oflabeled CFSs, along with a solution containing a reaction improvementanalogue; so that a complex A between said analyte and labeled CFS, anda complex B between said reaction improvement analogue and labeled CFSare formed by applying a voltage onto a capillary, arrangement order ofthese solutions may be determined, in accordance with arrangement orderof a solution containing an analyte or an analogue thereof, andsolutions including not less than one kind of CFSs, as explained above,by consideration on electrophoretic mobility of an analyte, a reactionimprovement analogue and a labeled CFS.

In addition, a solution containing an analyte or an analogue thereof,and solutions containing not less than one kind of CFSs are notnecessarily made adjacent, and liquid such as water, a physiologicalsalt solution, various buffer solutions, an organic solvent, and thelike may be inserted between these solutions. In this connection, assuch a buffer solution, any one not inhibit formation of a complexbetween an analyte or an analogue thereof and not less than one kind ofCFSs, can be used, including buffers usually used in this field, forexample, Tris buffer, Good's buffer, TE buffer, TAE buffer, TBE buffer,TBS buffer, a phosphate buffer, a borate buffer, and the like.

In this connection, a solution containing an analyte or an analoguethereof, and solutions including not less than one kind of CFSs, alongwith if necessary, the liquids, are formed and arranged as each separatezone in a capillary. In other words, between a solution containing ananalyte or an analogue thereof, and solutions including not less thanone kind of CFSs, along with if necessary, between the solutioncontaining an analyte or analogue thereof and the liquid or between thesolution including not less than one kind of CFSs and the liquid,liquid-liquid interface is formed and maintained at the time thesesolutions, and if necessary, the liquids are arranged.

In a step (1), as a method for introducing a solution containing ananalyte or an analogue thereof, and solutions including not less thanone kind of CFSs into a capillary, any method is allowed as long as azone of the solution containing an analyte or an analogue thereof, and azone of the solutions including not less than one kind of CFSs areseparately formed in a capillary, so that arrangement as described aboveis formed, in other words, any method is allowed as long asliquid-liquid interface is formable between the solution containing ananalyte or an analogue thereof, and the solutions including not lessthan one kind of CFSs, along with if necessary, between the solutioncontaining an analyte or analogue thereof and a liquid or between thesolution including not less than one kind of CFSs and the liquid, and awell-known methods for introducing can be used. Such well-known methodfor introducing includes, for example, a method for electricallyintroducing these solutions (and the liquids) into a capillary byapplying a voltage onto a capillary; a method for introducing thesesolutions (and the liquids) into a capillary increasing and/or reducinga pressure of the capillary inside; a method for introducing thesesolutions (and the liquids) into a capillary using capillary phenomenon;and the like.

In addition as a method for introducing and arranging a solutioncontaining an analyte or an analogue thereof, and solutions includingnot less than one kind of CFSs into a capillary, known methodsthemselves for introducing and arranging can be used. Such known methodfor introducing and arranging includes, for example, (1) a method forintroducing first one kind of a solution among a solution containing ananalyte or an analogue thereof, and solutions including not less thanone kind of CFSs into a capillary from the tip of a capillary, by amethod for introduction as described above, and subsequently introducingone kind of the remaining solutions into a capillary from the tip of acapillary similarly by a method for introduction as described above,which is repeated till all of the solutions are arranged into acapillary; (2) a method for dropping first one kind of a solution amonga solution containing an analyte or an analogue thereof, and solutionsincluding not less than one kind of CFSs into a fluid reservoir (awell), and introducing the content into a capillary by a method forintroduction as described above, and then replacing the solution in thefluid reservoir (the well) with one kind of a solution among theremaining solutions, and introducing the content into a capillary by amethod for introduction as described above, which is repeated till allof the solutions are arranged into a capillary; (3) a method fordropping a solution containing an analyte or an analogue thereof, andsolutions including not less than one kind of CFSs, separately into aplurality of fluid reservoirs (wells), and introducing these separatelyinto the same capillary by a method for introduction as described above,to arrange all of the solutions into a capillary; and the like. In thisconnection, a method for introducing a solution containing an analyte oran analogue thereof, and solutions including not less than one kind ofCFSs into a capillary, and a method for introducing and arranging asolution containing an analyte or an analogue thereof, and solutionsincluding not less than one kind of CFSs into a capillary are notlimited to the above-described methods.

In addition, a method for introducing the following: A sample containingan analyte; a solution including a sample having an analyte and alabeled analogue or a reaction improvement analogue (a solutioncontaining an analyte or an analogue); a solution containing an analyteand not less than one kind of CFSs (for example, a CFS, a labeled CFS, areaction improvement CFS, combinations thereof); a solution including alabeled analogue; or a solution including a reaction improvementanalogue; into a capillary is the same as a method for introducing asolution containing an analyte or an analogue thereof, and solutionsincluding not less than one kind of CFSs into a capillary, as describedabove.

1-2. A Step of Concentrating Reaction [a Step (2)]

A step (2) of the present invention is a step of contacting said analyteor analogue thereof and CFS while concentrating said analyte or analoguethereof and/or CFS electrophoretically to form a complex between saidanalyte or analogue thereof and CFS, not by (not depending on) moleculardiffusion.

“Before solutions (a solution containing an analyte or an analoguethereof and solutions containing not less than one kind of CFSs) areuniformly mixed” means “before each zone (liquid-liquid interface) of asolution containing an analyte or an analogue thereof and a solutionincluding not less than one kind of CFSs, along with if necessary theliquid, arranged into a capillary by a step (1) of the presentinvention, are uniformly mixed by molecular”.

In this connection, in the present invention, “interface” means,boundary where a solution containing an analyte or an analogue thereof,and solutions including not less than one kind of CFSs are contacting,or if necessary boundary where the solution containing an analyte oranalogue thereof and the liquid are contacting or boundary where thesolution including not less than one kind of CFSs and the liquid arecontacting, and said interface does not necessarily mean completely nomixing, due to presence of diffusion from a practical standpoint.

In addition, in a competitive method using an analogue of an analyte,and when a labeled analogue is used, the above-described term means“before uniformly mixing the following zones (liquid-liquid interface)arranged into a capillary by a step (1), by molecular diffusion”; (1) aneach zone (liquid-liquid interface) of a solution containing a samplehaving an analyte and a labeled analogue (a solution containing ananalyte and an analogue) along with a solution containing not less thanone kind of CFSs (a CFS, a reaction improvement CFS, combinationsthereof), and if necessary the liquid; (2) an each zone (liquid-liquidinterface) of a sample containing an analyte, a solution containing alabeled analogue and a solution containing not less than one kind ofCFSs (a CFS, a reaction improvement CFS, combinations thereof), and ifnecessary the liquid; or (3) an each zone (liquid-liquid interface) of asolution containing a sample having an analyte and not less than onekind of CFSs (a CFS, a reaction improvement CFS, combinations thereof)and a solution containing a labeled analogue, and if necessary theliquid.

In a competitive method using an analogue of analyte, when a reactionimprovement analogue is used, the above-described term means “beforeuniformly mixing the following zones (liquid-liquid interface) bymolecular diffusion”; (1) an each zone (liquid-liquid interface) of asolution containing a sample having an analyte and a reactionimprovement analogue (a solution containing an analyte and an analogue)along with a solution containing not less than one kind of labeled CFSs,and if necessary the liquid; (2) an each zone (liquid-liquid interface)of a sample containing an analyte, a solution containing a reactionimprovement analogue and a solution containing not less than one kind oflabeled CFSs, and if necessary the liquid; or (3) an each zone(liquid-liquid interface) of a solution containing a sample having ananalyte and not less than one kind of CFSs (a CFS, a labeled CFS,combinations thereof) and a solution containing a reaction improvementanalogue, and if necessary the liquid.

In this connection, “interface” also has the same meaning as above.

In a step (2), “concentrating an analyte or an analogue thereof, and/orat least one kind of a CFS by applying a voltage onto a capillary” meansthat at least one kind among an analyte or an analogue thereof and notless than one kind of CFSs gather in band-like (plug-like) onapplication of a voltage onto a capillary. In other words, it means thatsaid substances gather on application of a voltage onto a capillary sothat such a portion is generated wherein concentration of said substancebecomes higher than that of a substance in a zone arranged in a step(1), namely it means that an analyte or an analogue thereof and/or a CFSgather on application of a voltage onto a capillary, and a portion isgenerated wherein concentration of an analyte or an analogue thereofand/or concentration of a CFS becomes higher than that of an analyte oran analogue and/or that of a CFS in a solution zone (for example, a zoneof a solution containing an analyte or an analogue thereof, a zone of asolution containing a CFS) arranged in a step (1).

In this connection, as for level (degree) of concentration in thepresent invention, concentration of an analyte or an analogue thereofand/or at least one kind of a CFS at an gathered part (band-like) ofsaid substance on application of a voltage onto a capillary, relative toconcentration of an analyte or an analogue thereof and/or at least onekind of a CFS in a zone arranged by a step (1) is, as lower limit,usually not lower than 1.5 times, preferably not lower than 5 times,more preferably not lower than 10 times, and further preferably notlower than 25 times, and upper limit is not especially limited, howeverusually not higher than 10⁷ times, preferably not higher than 10⁶ timesand more preferably not higher than 10⁵ times.

In addition, in the present invention, “contacting said analyte oranalogue thereof with CFS” means that, as described above, contactbetween an analyte or an analogue thereof and a CFS is occurred not by(not depending on) molecular diffusion and by the phenomenon that asubstance with (having?) higher electrophoretic mobility (fasterelectrophoretic speed) among an analyte or an analogue thereof and notless than one kind of CFSs overtakes a substance with (having?) lowerelectrophoretic mobility (slow electrophoretic speed), by utilization ofthe fact that when electrophoresis carried out under the condition thata solution containing a substance with (having?) higher electrophoreticmobility (faster electrophoretic speed) is arranged at upstream of asolution containing a substance with (having?) lower electrophoreticmobility (slow electrophoretic speed), a substance with (having?) higherelectrophoretic mobility (faster electrophoretic speed) in a solutionovertakes a substance with (having?) lower electrophoretic mobility(slow electrophoretic speed).

Namely, in a step (2) of the present invention, an analyte or ananalogue thereof in a solution, and a CFS in a solution are made contactby movement (migration) electrophoretically, without mixing by(depending on) molecular diffusion generated by standing still asolution containing an analyte or an analogue thereof and a solutionincluding not less than one kind of CFSs, along with if necessary theliquid, arranged in a capillary, or also not by physical mixing thereofin a capillary.

As described above, a step (2) of the present invention forms a complexbetween said analyte or analogue thereof and CFS by migrating an analyteor analogue thereof in a solution and a CFS in a solutionelectrophoretically and contacting them while concentrating an analyteor analogue thereof and/or a CFS electrophoretically, before a solutioncontaining an analyte or an analogue thereof and solutions including aCFSs, along with if necessary the liquid, are uniformly mixed bymolecular diffusion, and further without uniformly mixing thesephysically in a capillary, in other words, with maintaining aliquid-liquid interface between these adjacent solutions and ifnecessary between the liquid and the solution.

In the present invention, “contacting said analyte or analogue thereofwith CFS while concentrating said analyte or analogue thereof and/or atleast one kind of CFS by applying a voltage to a capillary” means thefollowing both cases: (1) The case that the above-mentionedconcentration of an analyte or an analogue thereof and/or a CFS, andcontact of an analyte or an analogue thereof and a CFS aresimultaneously carried out; or (2) the case that after substantialcompletion of the above-mentioned concentration of an analyte or ananalogue thereof and/or a CFS, contact of an analyte or an analoguethereof and a CFS is carried out. Therefore, the term includes the casesother than the case that concentration of an analyte or an analoguethereof and/or a CFS is carried out after substantial completion ofcontact between an analyte or an analogue thereof and a CFS.

In other words, a step (2) of the present invention can be carried outby applying a voltage onto said capillary under condition that a complexbetween said analyte or analogue thereof and CFS can be formed bycontacting said analyte or analogue thereof and CFS while concentratingsaid analyte or analogue thereof and/or at least one kind of CFS asdescribed above by applying a voltage onto said capillary as describedabove, before a solution containing an analyte or an analogue thereofand solutions containing not less than one kind of CFSs are uniformlymixed.

Such conditions are specifically those used in so-called anelectrophoresis concentration method for concentration of substances ina capillary.

An electrophoresis concentration method includes, for example, methodsusing difference in electrophoretic mobility in a capillary such as (1)Field Amplification Sample Stacking Method (FASS) [US-A-2003-0057092 A1;Weiss, D. J., Saunders, K., Lunte, C. E. Electrophoresis 2001, 22,59-65; Britz-McKibbin, P., Bebault, G. M., Chen, D. D. Y. Anal Chem.2000, 72, 1729-1735; Ross, D., Locascio, L. E. Anal Chem. 2002, 71,5137-5145, and the like]; (2) Field Amplification Sample InjectionMethod (FASI) [Chien, R. L et al. J. Chromatogr. 1991, 559, 141-148, andthe like]; (3) Isotachophoresis (ITP) [Everaerts, F. M., Geurts, M.Mikkers, F. E. P., Verheggen, T. P. E. M J. Chromatogr. 1976, 119,129-155; Mikkers, F. F. P., Everaerts, F. M., Peek, J. A. F. J.Chromatogr. 1979, 168, 293-315; Mikkers, F. E. P., Everaerts, F. M.,Peek, J. A. F. J. Chromatogr. 1979, 168, 317-332; Hirokawa, T, Okamoto,H. Ikuta, N., and Gas, B., Analytical Sciences 2001, Vol. 17 Supplementil85, and the like]; (4) Isoelectric Focusing method (IF) [Wehr T, etal., Am. Biotechnol. Lab. 1990, 8, 22; Kilar F. et al., Electrophoresis1989, 10, 23-29, and the like]; (5) Large-volume sample stacking method(LVSS) [Siri, N. et al., J. Chormatogr. B, (2003), 793, 151-157, and thelike]; (6) pH junction method (pH-mediated stacking) [P. Britz-McKibbinet al., 2000, Anal. Chem., 72, 1242, P. Britz-McKibbin et al., 2002,Anal. Chem., 74, 3736]; (7) Sweeping method (stacking micellarelectrokinetic chromatography) [J. P. Quirino et al., 1998, Science,282, 465, J. P. Quirino et al., 1999, Anal. Chem., 71, 1638, Y. Sera etal., 2001, Electrophoresis, 22, 3509]; and the like, however, notlimited thereto.

Among the electrophoresis condensation methods described above, forexample, ITP, FASS are preferable, and ITP is particularly preferable.

In this connection, ITP is based on principle that an objectivesubstance can be concentrated when the objective substance is sandwichedbetween an electrophoresis medium (a leading buffer) including a leadingion having faster electrophoretic speed than that of an objectivesubstance, and an electrophoresis medium (a trailing buffer) including atrailing ion having slow electrophoretic speed than that of an objectivesubstance and subjected to electrophoresis.

Therefore, in carrying out a step (2) of the present invention by ITP,at least a an electrophoresis medium (leading buffer) including anleading ion having faster electrophoretic speed than that of an analyteor an analogue thereof and/or not less than one kind of CFSs, and anelectrophoresis medium (a trailing buffer) including a trailing ionhaving slow electrophoretic speed than that of an analyte or an analoguethereof and/or not less than one kind of CFSs are necessary, and thesecomponents-are also included in “condition that an analyte or ananalogue thereof and/or at least not less than one kind of CFSs areconcentrated” of the present invention.

In this connection, a leading buffer is arranged further downstream sideof a solution arranged at the most downstream side of a capillary, amonga solution containing an analyte or an analogue thereof, and solutionsincluding not less than one kind of CFSs, and a trailing buffer isarranged further upstream side of a solution arranged at the mostupstream side of a capillary, among a solution containing an analyte oran analogue thereof, and solutions including not less than one kind ofCFSs.

In the above description, as a leading ion, any ion having fasterelectrophoretic speed than that of an analyte or an analogue thereofand/or not less than one kind of CFSs may be used, and suitably beselected from those usually used in this field. Such ion includes, forexample, Cl⁻, and the like. In addition, use concentration of a leadingion may also suitably be selected from a range usually used in thisfield. Use concentration is, for example, usually 1 μM to 10 M,preferably 100 μM to 1 M, and more preferably 1 mM to 500 mM.

Also a leading buffer including such a leading ion is used by suitablyselected from one usually used in this field, and for example, Good'sbuffer, Tris buffer, a borate buffer, a phosphate buffer, a histidinebuffer, an imidazole buffer, a glycine buffer, and the like areincluded. Use concentration and pH thereof may suitably be selected fromthose usually used in this field, and use concentration is usually 1 μMto 10 M, preferably 100 μM to 1 M, and more preferably 1 mM to 500 mM,and pH is usually 2 to 12, preferably 4 to 10 and more preferably 6 to9.

In the above description, as a trailing ion, any ion having slowelectrophoretic speed than that of an analyte or an analogue thereofand/or not less than one kind of CFSs may be used, and suitably beselected from one usually used in this field. Such ion includes,including, for example, Good's buffer such as HEPES, TAPS, MES, MOPS,and the like, an amino acid of such as glycine, threonine and the like.In addition, use concentration of a trailing ion may also suitably beselected from a range usually used in this field. Use concentration is,for example, usually 1 μM to 10 M, preferably 100 μM to 1 M, and morepreferably 1 mM to 500 mM.

Also a trailing buffer including such a trailing ion is used by suitablyselected from one usually used in this field, and for example, Good'sbuffer, Tris buffer, a borate buffer, a phosphate buffer, a histidinebuffer, an imidazole buffer, a glycine buffer, and the like areincluded. Use concentration and pH thereof may suitably be selected fromthose usually used in this field, and use concentration is usually 1 μMto 10 M, preferably 100 μM to 1 M, and more preferably 1 mM to 500 mM,and pH is usually 2 to 12, preferably 4 to 10 and more preferably 6 to9.

In this connection, the above-described conditions (a leading ion, aleading buffer, a trailing ion, a trailing buffer, and the like), otherreagents, operation methods, other conditions, and the like can beselected according to the description in the above-described references,and the like.

FASS, FASI and LVSS are based on principle that electrophoretic mobilityof an objective substance is decreased and an objective substance isconcentrated, when an objective substance reaches interface between amedium wherein an objective substance is present, and a medium havinghigher electric conductivity than that of a medium wherein an objectivesubstance is present.

Therefore, when a step (2) of the present invention is carried out byFASS, LVSS or FASI, at least one kind of a solution among a solutioncontaining an analyte or an analogue thereof, and solutions includingnot less than one kind of CFSs is required to have higher electricconductivity than that of other at least one kind of a the solutions; oran electrophoresis medium (high electric conductivity electrophoresismedium) having higher electric conductivity than that of a solutioncontaining an analyte or an analogue thereof, and/or solutions includingnot less than one kind of CFSs is required to be separately used, andthese components are also included in “condition that an analyte or ananalogue thereof and/or not less than one kind of CFSs are concentrated”of the present invention.

In this connection, a high electric conductivity electrophoresis mediumis arranged at further downstream side of a solution arranged at themost downstream in a capillary, among a solution containing an analyteor an analogue thereof, and solutions including not less than one kindof CFSs.

In addition, a high electric conductivity electrophoresis medium may beany one as long as having higher salt concentration than that of asolution containing an analyte or an analogue thereof, and/or solutionsincluding not less than one kind of CFSs, and is used by suitably beselected from one usually used in this field. Such high electricconductivity electrophoresis medium includes, for example, anelectrophoresis medium containing NaCl, KCl, and the like. As anelectrophoresis medium, for example, Good's buffer, Tris buffer, aborate buffer, a phosphate buffer, a histidine buffer, an imidazolebuffer, a glycine buffer, and the like are included. Use concentrationand pH thereof may suitably be selected from those usually used in thisfield, and use concentration is usually 1 μM to 10 M, preferably 100 μMto 1 M, and more preferably 1 mM to 500 mM, and pH is usually 2 to 12,preferably 4 to 10 and more preferably 6 to 9.

In this connection, the above-described conditions (high electricconductivity electrophoresis medium, and the like), other reagents,operation methods, other conditions, and the like can suitably beselected according to the description in the above-described references,and the like.

IF is based on principle that an objective substance is concentrated byfilling a capillary (channel) with a solution of amphoteric substanceshaving various isoelectric points, then forming pH gradient in acapillary (channel) by applying a voltage, and when an objectivesubstance reaches pH region corresponding to an isoelectric point.

Therefore, in the case when a step (2) of the present invention iscarried out by IF, at least an electrophoresis medium formable pHgradient in a capillary is required, and this component is also includedin “condition to make an analyte or an analogue thereof and/or at leastone kind of CFSs concentrated” of the present invention.

In the above description, as an electrophoresis medium formable pHgradient in a capillary, anyone formable pH gradient in a capillary byapplying a voltage is used by suitably selecting from one usually usedin this field. As such an electrophoresis medium formable pH gradient ina capillary, for example, an electrophoresis medium containing asubstance formable pH gradient in a capillary, such as ampholyte isincluded. As such an electrophoresis medium, for example, Good's buffer,Tris buffer, a borate buffer, a phosphate buffer, a histidine buffer, animidazole buffer, a glycine buffer, and the like are included. Useconcentration and pH thereof also may suitably be selected from thoseusually used in this field, and use concentration is usually 1 μM to 10M, preferably 100 μM to 1 M, and more preferably 1 mM to 500 mM.

In this connection, the above-described conditions (an electrophoresismedium containing a substance formable pH gradient in a capillary, andthe like), other reagents, operation methods, other conditions, and thelike can be selected according to the description in the above-describedreferences, and the like.

A pH junction method is one for carrying out concentration of anobjective substance contained in a sample at the boundary surfacebetween a sample and an alkaline electrophoresis medium by forming anacidic or a weak acidic sample region (zone) in the alkalineelectrophoresis medium.

Therefore, in the case when a step (2) of the present invention iscarried out by a pH junction method, at least an electrophoresis mediumhaving more alkaline range pH than that of a solution containing asample is required, and this component is also included in “condition tomake an analyte or an analogue thereof and/or not less than one kind ofCFSs concentrated” of the present invention.

In the above description, as an electrophoresis medium having alkalinerange pH, any one formable a boundary surface with different pH, betweena solution containing a sample and said electrophoresis medium in acapillary by applying a voltage is used by suitably selected from oneusually used in this field. As such an electrophoresis medium, forexample, Good's buffer such as HEPES, TAPS, MES, MOPS, and the like, aborate buffer, a phosphate buffer, a histidine buffer, an imidazolebuffer, a glycine buffer, and the like are included. Use concentrationand pH thereof may suitably be selected from those usually used in thisfield, and use concentration is usually 1 μM to 10 M, preferably 100 μMto 1 M, and more preferably 1 mM to 500 mM, and pH is usually 7 to 11,preferably 7 to 10 and more preferably 7 to 9.

In this connection, the above-described conditions (an electrophoresismedium having alkaline range pH, and the like), other reagents,operation methods, other conditions, and the like can be selectedaccording to the description in the above-described references, and thelike.

A sweeping method is based on the following principle:

Namely, an electrophoresis medium including a charged substance forminga micelle is arranged at the upstream side of a solution zone includingan objective substance. By applying a voltage here, the micelle formedovertakes an objective substance and forms a micelle complex with anobjective substance. When the micelle complex reaches interface betweena medium wherein an objective substance is present, and a medium havinghigher electric conductivity than that of a medium wherein an objectivesubstance is present, electrophoretic speed of an objective substance isdecreased and thus an objective substance is concentrated.

Therefore, when a step (2) of the present invention is carried out by asweeping method, at least one kind of a solution among a solutioncontaining an analyte or an analogue thereof and solutions including notless than one kind of CFSs is required to have high electricconductivity than that of other at least one kind of a the solutions; oran electrophoresis medium (high electric conductivity electrophoresismedium) having higher electric conductivity than that of a solutioncontaining an analyte or an analogue thereof and/or solutions includingnot less than one kind of CFSs is required to separately be used; and anelectrophoresis medium including a charged substance forming a micellewith higher electrophoretic speed than that of an analyte or an analoguethereof and/or not less than one kind of CFSs, and having lower electricconductivity than that of a solution (or an electrophoresis medium)having higher electric conductivity is required; and these componentsare also included in “condition to make an analyte or an analoguethereof and/or not less than one kind of CFSs concentrated” of thepresent invention.

In this connection, a high electric conductivity electrophoresis mediumis arranged further downstream side of a solution arranged at the mostdownstream side of a capillary, among a solution containing an analyteor an analogue thereof, and solutions including not less than one kindof CFSs, and an electrophoresis medium including a charged substanceforming a micelle is arranged further upstream side of a solutionarranged at the most upstream side of a capillary, among a solutioncontaining an analyte or an analogue thereof, and solutions includingnot less than one kind of CFSs.

In the above description, as a charged substance forming a micelle, anycharged substance may be used as long as having faster electrophoreticspeed than that of an analyte or an analogue thereof and/or not lessthan one kind of CFSs, and suitably be selected from one usually used inthis field. Such charged substance includes, for example, a surfactantsuch as SDS, and the like. In addition, use concentration of saidcharged substance may also suitably be selected from a range usuallyused in this field, and the amount over critical micelle concentrationis used, and, in more specifically, use concentration of said chargedsubstance is for example, usually 1 μM to 10 M, preferably 100 μM to 1M, and more preferably 1 mM to 500 mM.

Also an electrophoresis medium is used by suitably selected from oneusually used in this field, and, for example, Good's buffer, Trisbuffer, a borate buffer, a phosphate buffer, a histidine buffer, animidazole buffer, a glycine buffer, and the like are included. Useconcentration and pH thereof may suitably be selected from those usuallyused in this field, and use concentration is usually 1 μM to 10 M,preferably 100 μM to 1 M, and more preferably 1 mM to 500 mM, and pH isusually 2 to 12, preferably 4 to 10 and more preferably 6 to 9.

In this connection, the above-described conditions [a charged substanceforming a micelle, an electrophoresis medium, and the like], otherreagents, operation methods, other conditions, and the like can suitablybe selected according to the description in the above-describedreferences, and the like.

Applied voltage in a step (2) may be in a range wherein an analyte oranalogue thereof and/or a CFS is sufficiently concentrated, and acomplex between an analyte or an analogue thereof and a CFS issufficiently formed, and may suitably be selected from that usually usedin this field. In more specifically, voltage is applied, so thatelectric field intensity is in the following range: as lower limit,usually not lower than 5 V/cm, preferably not lower than 10 V/cm, morepreferably not lower than 50 V/cm, further preferably not lower than 500V/cm, and particularly preferably not lower than 1000 V/cm, and as upperlimit, usually not higher than 10000 V/cm, preferably not higher than5000 V/cm, more preferably not higher than 2000 V/cm.

In addition, other reaction conditions (for example, pH, temperature,time, and the like) preferably be in a range not to inhibitconcentration of an analyte or analogue thereof and/or a CFS, andformation of a complex between an analyte or an analogue thereof and aCFS.

Specifically, although not to simply be described because of dependencyon property of an analyte or an analogue thereof and a CFS, lower limitof pH is usually not lower than 2, preferably not lower than 5, andupper limit of pH is usually not higher than 10, and preferably nothigher than 9; and lower limit of temperature is usually not lower than0° C., preferably not lower than 5° C., and more preferably not lowerthan 10° C., and upper limit of temperature is usually not higher than50° C., preferably not higher than 40° C., and more preferably nothigher than 30° C. Reaction time depends on binding constant of a CFS tobe used to an analyte or an analogue thereof, and relatively low bindingconstant requires relatively long reaction time but relatively highbinding constant requires relatively short reaction time. In morespecifically, for example, lower limit is usually not shorter than 1minute, preferably not shorter than 3 minutes and more preferably notshorter than 5 minutes; and upper limit is usually not longer than 24hours, preferably not longer than 12 hours, more preferably not longerthan 1 hour and further preferably not longer than 30 minutes.

1-3. A Capillary (Channel)

A capillary (channel) used in the present invention is any one usuallyused in this field such as a capillary electrophoresis method, acapillary chip electrophoresis method, and the like, and not especiallylimited.

Material of a capillary (channel) used in the present invention is anyone usually used in this field, and not especially limited as long as itcan finally form a complex between an analyte or an analogue thereof andnot less than one kind of CFSs by making contact of an analyte or ananalogue thereof and not less than one kind of CFSs. Examples of amaterial of capillary (channel) are, for example, silica-based compoundssuch as glass, quartz and silicon, synthetic polymers such as cyclicolefin copolymer (COC), cyclic olefin polymer (COP),polymethylmethacrylate, polymethylsiloxane, polyvinyl chloride,polyurethane, polystyrene, polysulfone, polycarbonate,polytetrafluoroethylene, and the like. In addition, inner diameter andlength of a capillary (channel) are not especially limited as long asthey are sufficient to concentrate an analyte or an analogue thereofand/or a CFS, and form a complex between an analyte or analogue thereofand a CFS. For example, inner diameter is usually 1 to 1000 μm,preferably 1 to 200 μm and more preferably 1 to 100 μm, and length isusually 0.1 mm to 100 cm, preferably 0.1 mm to 20 cm and more preferably0.1 mm to 10 cm.

A step (2) of the present invention is usually carried out in a statethat the above-mentioned capillary is filled with an electrophoresismedium. As an electrophoresis medium, a buffer solution forelectrophoresis or said buffer solution for electrophoresis containingfillers, and the like are included. In this connection, anelectrophoresis medium may be used alone or in combination with two ormore kinds. In addition, as a method for introducing an electrophoresismedium into a capillary includes a method for introducing a solutioncontaining an analyte or an analogue thereof, and solutions includingnot less than one kind of CFSs into a capillary as described above. Andany one of the following timings of introduction of an electrophoresismedium into a capillary may be adopted: (1) before the introduction of asolution containing an analyte or an analogue thereof, and/or solutionsincluding not less than one kind of CFSs into a capillary; (2)simultaneously with the introduction of a solution containing an analyteor an analogue thereof, and/or solutions including not less than onekind of CFSs into a capillary; and (3) after the introduction of asolution containing an analyte or an analogue thereof, and/or solutionsincluding not less than one kind of CFSs into a capillary.

Such a buffer solution for electrophoresis is not especially limited aslong as it is usually used in this field. Examples of a buffer solutionfor electrophoresis are, for example, the buffer solutions etc. usuallyused in the field of a hybridization method, an immunization, and thelike such as Tris buffer, a phosphate buffer, Veronal buffer, a boratebuffer, Good's buffer, SSC buffer, TBE buffer, TAE buffer, and the like.Concentration of these buffer solutions is usually 0.1 mM to 10 M,preferably 1 mM to 5 M, and more preferably 5 mM to 1M. In addition, anypH of said buffer solution may be used as long as not to give bad effecton separation of substances, and is usually 2 to 13, preferably 4 to 11and more preferably 5 to 9. In this connection, these buffer solutionsmay be used alone or in combination with two or more kinds.

Fillers (polymers) filled in a capillary are not especially limited aslong as they are usually used in this field. Examples of fillers are,for example, polyethers such as polyethylene oxide (polyethyleneglycol), polypropylene oxide; polyalkylene imines such as polyethyleneimine; polyacrylic acid polymers such as polyacrylic acid, polyacrylateesters, and poly(methyl acrylate); polyamide-based polymers such aspolyacrylamide, polymethacrylamide; polymethacrylic acid-based polymerssuch as polymethacrylic acid, polymethacrylate esters and poly(methylmethacrylate); polyvinyl-based polymers such as polyvinyl acetate,polyvinyl pyrrolidone and polyvinyl oxazolidone; water-soluble hydroxylpolymers such as pullulan, elsinan, xanthan, dextran and guar gum;water-soluble cellulosic compounds such as methyl cellulose,hydroxyethyl cellulose and hydroxypropyl cellulose; derivatives thereof,and copolymers having a plurality of kinds of monomer unites composingthese polymers, and the like. In this connection, these fillers may beused alone or in combination with two or more kinds.

Molecular weight of the fillers as described above is usually 500 Da to6000 kDa, preferably 1 to 1000 kDa and more preferably 50 to 500 kDa.

Use concentration of the fillers as described above may suitably beselected from a range usually used in this field, and is usually 0.01 to40% (w/v), preferably 0.01 to 20% (w/v), and more preferably 0.1 to 10%(w/v).

In this connection, viscosity of a buffer solution for electrophoresiswhen the above-described fillers are added thereto, is usually 1 to 1000centipoises, preferably 1 to 200 centipoises, and more preferably 1 to10 centipoises.

1-4. Analyte, Sample, Analogue and Solution Containing Analyte orAnalogue

(1) Analyte

An analyte in the present invention includes, for example, a nucleotidechain (an oligonucleotide chain and a polynucleotide chain, etc.); achromosome; a peptide chain (C-peptide and angiotensin I, etc.), protein[procalcitonin, immunoglobulin A (IgA), immunoglobulin E (IgE),immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin D (IgD),β₂-microglobulin, albumin, decomposition product thereof, and serumprotein such as ferritin, etc.]; enzyme [an amylase (pancreatic type,salivary gland type and X-type, etc.), an alkaline phosphatase (hepatic,osseous, placental and small intestinal, etc.), an acid phosphatase(PAP, etc.), a γ-glutamyl transferase (renal, pancreatic and hepatic,etc.), a lipase (pancreatic type and gastric type, etc.), a creatinekinase (CK-1, CK-2 and mCK, etc.), a lactate dehydrogenase (LDH1 toLDH5, etc.), a glutamate oxaloacetate transaminase (ASTm and ASTs,etc.), a glutamate-pyruvate transaminase (ALTm and ALTs, etc.), acholine esterase (ChE1 to ChE5, etc.), a leucine aminopeptidase (C-LAP,AA and CAP, etc.), renin, a protein kinase and a tyrosine kinase, etc.];microorganism such as bacteria (tuberculosis bacteria, pneumococcalorganisms, diphtheria organisms, meningococcus, gonococcus,staphylococcus, streptococcus, enteric bacteria, coliform bacillus andHelicobacter pylori, etc.), viruses (rubella virus, herpes virus,hepatitis virus, ATL virus, AIDS virus, influenza virus, adenovirus,enterovirus, poliovirus, EB virus, HAV, HBV, HCV, HIV and HTLV, etc.),fungus (candida and Cryptococcus, etc.), spirochete (leptospire,Treponema pallidum, etc.), chlamydia and mycoplasma; protein, a peptideor a carbohydrate antigen derived from said microorganisms; variousallergen causative of bronchospasm, allergic rhinitis and atopicdermatitis, etc. (allergen derived from the house dust, mites such asDermatophagoides farinae and Dermatophagoides pteronyssinus, etc.,pollen from cedar, cypress, Pasupalum thunbergii, ragweed, timothy,sweet vernal grass and rye, etc., an animal such as a cat, a dog or acrab, etc., food such as rice and egg white, etc., fungus, insect, wood,drug or chemical substance, etc.); lipids (lipoprotein, etc.); protease(trypsin, plasmin and serine protease, etc.); protein antigen tumormarker (PSA, PGI and PGII, etc.); a carbohydrate antigen [AFP (L1 to L3,etc.), hCG (hCG family, etc.), transferrin, IgG, thyroglobulin,Decay-accelerating factor (DAF), carcinoembryonic antigen (CEA, NCA,NCA-2 and NFA, etc.), CA19-9, PIVKA-II, CA125, prostate-specificantigen, a carbohydrate antigen tumor marker having a particularcarbohydrate (sugar) chain produced by cancer cell and an ABOcarbohydrate antigen, etc.]; carbohydrate (sugar) chain [hyaluronicacid, β-glucan and carbohydrate (sugar) chain contained in theabove-described carbohydrate antigen, etc.]; a carbohydrate (sugar)chain binding protein (hyaluronic acid binding protein and β-glucanbinding protein, etc.); lectin (concanavalin A, lentil lectin, kidneybean lectin, thorn apple lectin and wheat germ lectin, etc.);phospholipid (cardiolipin, etc.); lipopolysaccharide (endotoxin, etc.);chemical substances (hormones such as PTH, T3, T4, TSH, insulin, LH, FSHand prolactin, etc., an endocrine-disturbing chemicals such astributyltin, nonyl phenol, 4-octyl phenol, di-n-butyl phthalate,dicyclohexyl phthalate, benzophenone, octachlorostyrene anddi-2-ethylhexyl phthalate, etc.); a receptor (receptor for estrogen andTSH, etc.); a ligand (estrogen and TSH, etc.); and antibodies thereto,and the like.

Among the above-described, the method of the present invention is usefulfor analysis (quantitative determination) of glycoprotein havingdifferent carbohydrate (sugar) chain structure, a nucleotide chain(oligonucleotide chain and polynucleotide chain) and a peptide chain(including polypeptide), is especially useful for glycoprotein havingdifferent carbohydrate (sugar) chain structure. In this connection, asthe carbohydrate (sugar) chain structure is known to be changed in aparticular disease such as cancer, and reported its usefulness in theclinical laboratory (clinical chemistry), the glycoprotein havingdifferent carbohydrate (sugar) chain structure can be reassessed for itsusefulness in the clinical laboratory (clinical chemistry) using themethod of the present invention. In addition, the separation of a mutanttype generated by minute mutation or substitution, etc. of a nucleotidechain (an oligonucleotide chain or a polynucleotide chain) or a peptidechain (including polypeptide), etc. from the wild type has beenconsidered as an important analytical target in the field of molecularbiology and molecular clinical laboratory (clinical chemistry), andtherefore, by analyzing (determining quantity) these mutant type and/orwild type using the method of the present invention, the possibility offinding out some valuable factors and the like in the clinicallaboratory (clinical chemistry) will be increased.

(2) Sample Containing Analyte

A sample containing an analyte of the present invention described abovemay be exemplified by the followings: samples derived from biologicalorigin including body fluid such as serum, plasma, spinal fluid,synovial fluid, lymph fluid, etc., excretions such as urine, faces,etc., expectoration, purulent matter, dermal exfoliation, environmentalsamples such as food, beverage, tapwater, seawater, waterof lakesandmarshes, river water, factory waste water, washings forsemiconductors, washings after washing of medical instruments, etc.; andtheir processed products reconstituted by dissolving in water or abuffer usually used in this field such as Tris buffer, phosphate buffer,Veronal buffer, borate buffer, Good's buffer, etc. In this connection, asample relevant to the present invention encompasses one containing ananalytes as described above produced by chemical synthesis.

(3) Analogue (a Labeled Analogue and a Reaction Improvement Analogue)

An analogue used in the present invention is a substance to which a CFS,binding to a target analyte for analysis in a sample, is bindable. Inother words, an alalogue is a substance a having the same binding siteas the binding site present in an analyte in said sample to which a CFSis bindable.

Such a substance includes, for example, the same one as an analyte in asample, a target of an analysis; one wherein a part of structure of ananalyte in a sample is modified, altered, denatured, removed, etc.(so-called an analogue); and the like. Examples of a substance are, forexample, recombinant protein introduced with partial mutation at ananalyte in a sample, a target of an analysis; peptide with partiallymodified peptide sequence of an analyte in a sample, a target of ananalysis; a nucleotide chain with partially modified nucleotide sequenceof an analyte in a sample, a target of an analysis; and the like. Inthis connection, specific examples of an analyte in a sample, a targetof an analysis, are as described above.

In this connection, a labeled analogue and a reaction improvementanalogue used in the present invention are ones wherein a labelingsubstance or a reaction improvement substance is bound to the abovedescribed substances, and specific examples and preferable embodimentsof a labeling substance and a reaction improvement substance are asdescribed later. In addition, a method for binding a labeling substanceor a reaction improvement substance to the above-described substancesmay be in accordance with a similar method to a method for bindingbetween a reaction improvement substance and a CFS or a method forlabeling a CFS by a labeling substance, to be described later.

Use amount of an analogue (a labeled analogue or a reaction improvementanalogue) is not simply described because of dependency on kinds of ananalogue (a labeled analogue or a reaction improvement analogue) to beused, or kinds or use concentration of a CFS, and the like.

In more specifically, an analogue (a labeled analogue or a reactionimprovement analogue) may be contained in a solution (for example, asolution containing an analyte and an analogue, a solution containing ananalogue, a solution containing an analogue and a CFS), so that useamount of an analogue (a labeled analogue or a reaction improvementanalogue) in a solution as described above is, as lower limit, usuallynot lower than 10 pM, preferably not lower than 1 nM and more preferablynot lower than 100 nM, and as an upper limit, usually not higher than 10μM, preferably not higher than 1 μM and more preferably not higher than500 nM.

(4) Solution Containing Analyte or Analogue

“A solution containing an analyte or an analogue” in the presentinvention means a solution including an analyte (a sample including theanalyte) or an analogue (a labeled analogue or a reaction improvementanalogue) of the present invention, as described above.

Such a solution includes (a) a sample itself including an analyte, asdescribed above, (b) a solution including a sample having an analyte andnot less than one kind of CFSs (in other words, a solution including acomplex between an analyte and not less than one kind of CFSs), (c) asolution containing an analogue (a labeled analogue or a reactionimprovement analogue), (d) a solution containing a sample having ananalyte and an analogue (a labeled analogue or a reaction improvementanalogue) (in other words, a solution including an analyte and ananalogue), (e) a solution containing an analogue (a labeled analogue ora reaction improvement analogue) and not less than one kind of CFSs (inother words, a solution including a complex between an analogue and notless than one kind of CFSs), (f) a solution including a sample having ananalyte, an analogue (a labeled analogue or a reaction improvementanalogue) and not less than one kind of CFSs, and the like.

In this connection, as the above solution (b) or (e), for example, when2 or more kinds of CFSs are used in the present invention, a solutioncontaining a complex (an intermediate complex) between a part of CFSsamong all of CFSs finally binding with an analyte or an analogue thereof(a part of the whole CFSs), and an analyte or an analogue thereof, inother words, a solution containing a complex (an intermediate complex)between CFS (s) fewer than CFSs composing a finally formed complexbetween an analyte or an analogue thereof and 2 or more kinds of CFSs,and an analyte or an analogue thereof is specifically included.

Namely, for example, when 2 kinds of CFSs are used, it is a solutioncontaining a complex (an intermediate complex) between one kind of a CFSand an analyte or an analogue thereof. And for example, when 3 kinds ofCFSs are used, it is a solution containing a complex (an intermediatecomplex) between one kind of a CFS and an analyte or an analoguethereof, and a solution containing a complex (an intermediate complex)between 2 kinds of CFSs and an analyte or an analogue thereof. (In thiscase, also when 4 or more kinds of CFSs are used, a way of thinking isthe same as in theses cases.)

In more specifically, for example, as will be described later, when aCFS not bound with a labeling substance and a reaction improvementsubstance, and a reaction improvement CFS are used in combination as aCFS, a solution containing a complex (an intermediate complex) betweenan analyte and said CFS, and a solution containing a complex (anintermediate complex) between an analyte and a reaction improvement CFScorrespond to a solution (b) as described above; and when a labeled CFSand a reaction improvement CFS are used in combination as a CFS, asolution containing a complex (an intermediate complex) between ananalyte and a labeled CFS, and a solution containing a complex (anintermediate complex) between an analyte and a reaction improvement CFScorrespond to a solution (b) as described above. In addition, when a CFSnot bound with a labeling substance and a reaction improvementsubstance, and a labeled reaction improvement CFS are used incombination as a CFS, a solution containing a complex (an intermediatecomplex) between an analyte and said CFS, and a solution containing acomplex (an intermediate complex) between an analyte and a labeledreaction improvement CFS correspond to a solution (b) as describedabove.

In this connection, in the above description, usually, a solutioncontaining a sample having an analyte, and not less than one kind ofCFSs is generally a reaction solution obtained by suitably mixing asample including an analyte as described above (a CFS is not included)and a solution including a CFS as will be described later.

The above solution (e) includes, in more specifically, for example, whena labeled analogue or a reaction improvement analogue is used, and when2 or more kinds of CFSs are used, a solution containing, similarly asdescribed above, a complex (an intermediate complex) between a part ofCFSs among all of CFSs finally binding with a labeled analogue or areaction improvement analogue (a part of the whole CFSs), and a labeledanalogue or a reaction improvement analogue, in other words, a solutioncontaining a complex (an intermediate complex) between CFS (s) fewerthan CFSs composing a finally formed complex between a labeled analogueor a reaction improvement analogue and 2 or more kinds of CFSs, and alabeled analogue or a reaction improvement analogue. In this connection,in the above description, usually, a solution containing an analogue (alabeled analogue or a reaction improvement analogue) is generally areaction solution obtained by suitably mixing a solution including alabeled analogue or a reaction improvement analogue, and a solutionincluding a CFS as will be described later.

In addition, as the above solution (d), for example, a solutioncontaining an analyte in a sample, and an analogue labeled with alabeling substance (a labeled analogue) or an analogue bound with areaction improvement substance (a reaction improvement analogue) isincluded. In this connection, a solution containing a sample having ananalyte and an analogue is usually and generally a mixed solutionobtained by suitably mixing a sample including an analyte (a CFS is notcontained) and a solution including an analogue.

As the above solution (f), in more specifically, for example, when alabeled analogue or a reaction improvement analogue is used, and when 2or more kinds of CFSs are used, a solution containing, similarly asdescribed above, a complex (an intermediate complex) between a part ofCFSs among all of CFSs finally binding with an analyte and/or a labeledanalogue (or a reaction improvement analogue), and an analyte and/or alabeled analogue (or a reaction improvement analogue) (a part of thewhole CFSs), in other words, a solution containing a complex (anintermediate complex) between CFS(s) fewer than CFSs composing a finallyformed complex between an analyte and/or a labeled analogue (or areaction improvement analogue) and 2 or more kinds of CFSs, and ananalyte and/or a labeled analogue (or a reaction improvement analogue).In this connection, a solution including a sample having an analyte, ananalogue (a labeled analogue or a reaction improvement analogue) and notless than one kind of CFSs is usually and generally a reaction solutionobtained by suitably mixing a solution including a sample having ananalyte as described above, a solution containing a labeled analogue ora reaction improvement analogue, and solutions including not less thanone kind of CFSs.

In the above description, (a) a sample including an analyte is asdescribed above. In addition, as solutions (b) to (f), any one is usedas long as not to inhibit formation of a complex between an analyte anda CFS, and/or a complex between an analogue (a labeled analogue or areaction improvement analogue) and a CFS, and for example, water, abuffer solution, and the like are included.

As such a buffer solution, one having buffer action at a pH range ofusually 5 to 11, and usually used in this field is included. Examples ofthe buffer solution include Tris buffer, Good's buffer, TE buffer, TAEbuffer, TBE buffer, TBS buffer, a phosphate buffer, a borate buffer, andthe like, and use concentration thereof is usually in a range of 1 mM to2 M, and preferably 10 mM to 1 M, and pH is usually 5 to 11, preferably5 to 10, more preferably 5.5 to 8.5, further preferably 6 to 8 andparticularly preferably around 7.

1-5. CFS and a Solution Containing Thereof

In the present invention, “a substance formable a complex with ananalyte or an analogue thereof (CFS)” means a substance having aproperty capable of forming a complex between a analyte or an analoguethereof and the CFS, namely, a complex containing the analyte or theanalogue thereof and the CFS as constituent by binding with an analyteor the analogue thereof as the above described, or by binding with theanalyte or the analogue thereof through other CFS.

Such CFS means the substance which binds with an analyte or an analoguethereof by interaction such as an “antigen”-“antibody” reaction, a“carbohydrate (sugar) chain”-“protein” reaction, “carbohydrate (sugar)chain”-“lectin” reaction, “enzyme”-“inhibitor” reaction,“protein”-“peptide chain” reaction or “chromosome or nucleotidechain”-“nucleotide chain” reaction, and the like. When one of thesubstances in the above-mentioned pairs is the analyte or the analoguethereof, the other is the CFS. For example, when an analyte or ananalogue thereof is an “antigen”, a CFS is an “antibody”, and when ananalyte or an analogue thereof is an “antibody”, a CFS is an “antigen”(the same applied to the above other pairs).

In more specifically, such a substance includes, for example, anucleotide chain (an oligonucleotide chain and a polynucleotide chain,etc.); a chromosome; a peptide chain (C-peptide and angiotensin I,etc.), protein [procalcitonin, immunoglobulin A (IgA), immunoglobulin E(IgE), immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin D(IgD), β₂-microglobulin, albumin, decomposition product thereof, andserum protein such as ferritin, etc.]; enzyme [an amylase (pancreatictype, salivary gland type and X-type, etc.), an alkaline phosphatase(hepatic, osseous, placental and small intestinal, etc.), an acidphosphatase (PAP, etc.), a γ-glutamyl transferase (renal, pancreatic andhepatic, etc.), a lipase (pancreatic type and gastric type, etc.), acreatine kinase (CK-1, CK-2 and mCK, etc.), alactate dehydrogenase (LDH1to LDH5, etc.), a glutamate oxaloacetate transaminase (ASTm and ASTs,etc.), a glutamate-pyruvate transaminase (ALTm and ALTs, etc.), acholine esterase (ChE1 to ChE5, etc.), a leucine aminopeptidase (C-LAP,AA and CAP, etc.), renin, a protein kinase and a tyrosine kinase, etc.];microorganism such as bacteria (tuberculosis bacteria, pneumococcalorganisms, diphtheria organisms, meningococcus, gonococcus,staphylococcus, streptococcus, enteric bacteria, coliform bacillus andHelicobacter pylori, etc.), viruses (rubella virus, herpes virus,hepatitis virus, ATL virus, AIDS virus, influenza virus, adenovirus,enterovirus, poliovirus, EB virus, HAV, HBV, HCV, HIV and HTLV, etc.),fungus (candida and Cryptococcus, etc.), spirochete (leptospire,Treponema pallidum, etc.), chlamydia and mycoplasma; protein, a peptideor a carbohydrate antigen derived from said microorganisms; variousallergen causative of bronchospasm, allergic rhinitis and atopicdermatitis, etc. (allergen derived from the house dust, mites such asDermatophagoides farinae and Dermatophagoides pteronyssinus, etc.,pollen from cedar, cypress, Pasupalum thunbergii, ragweed, timothy,sweet vernal grass and rye, etc., an animal such as a cat, a dog or acrab, etc., food such as rice and egg white, etc., fungus, insect, wood,drug or chemical substance, etc.); lipids (lipoprotein, etc.); protease(trypsin, plasmin and serine protease, etc.); antigen of tumor markerprotein (protein antigen of tumor marker?) (PSA, PGI and PGII, etc.); acarbohydrate antigen [AFP (L1 to L3, etc.), hCG (hCG family, etc.),transferrin, IgG, thyroglobulin, Decay-accelerating factor (DAF),carcinoembryonic antigen (CEA, NCA, NCA-2 and NFA, etc.), CA19-9,PIVKA-II, CA125, prostate-specific antigen, a carbohydrate antigen tumormarker having a particular carbohydrate (sugar) chain produced by cancercell and an ABO carbohydrate antigen, etc.]; carbohydrate (sugar) chain[hyaluronic acid, β-glucan and carbohydrate (sugar) chain contained inthe above-described carbohydrate antigen, etc.]; a carbohydrate (sugar)chain binding protein (hyaluronic acid binding protein and β-glucanbinding protein, etc.); lectin (concanavalin A, lentil lectin, kidneybean lectin, thorn apple lectin and wheat germ lectin, etc.);phospholipid (cardiolipin, etc.); lipopolysaccharide (endotoxin, etc.);chemical substances (hormones such as PTH, T3, T4, TSH, insulin, LH, FSHand prolactin, etc., an endocrine-disturbing chemicals such astributyltin, nonyl phenol, 4-octyl phenol, di-n-butyl phthalate,dicyclohexyl phthalate, benzophenone, octachlorostyrene anddi-2-ethylhexyl phthalate, etc.); a receptor (receptor for estrogen andTSH, etc.); a ligand (estrogen and TSH, etc.); and antibodies thereto,and the like. In this connection, the antibody used in the presentinvention encompasses a decomposition product such as Fab and F(ab′)₂fragments and the like produced by degradation with a proteolytic enzyme(proteinase, etc.) such as papain or pepsin or by chemical degradation.

The CFS described above may be used alone or in combination with two ormore kinds.

In this connection, when two or more kinds of the CFSs are used incombination (together), the binding site of each CFS is not especiallylimited as long as two or more kinds of the CFSs can form a complex withan analyte or an analogue thereof. The binding sites to be bound by suchCFSs include, for example, a case when all of binding sites to be boundby two or more of the CFSs are present on the analyte or the analoguethereof [binding form (1)]; a case when the binding site to be bound byat least one kind of the CFS (for example, CFS A) among two or more ofthe CFSs is present only on the analyte or the analogue thereof, and thebinding site to be bound by another at least one kind of the CFS (forexample, complex binding substance B) is present on a site newlygenerated by the formation of a complex between an analyte or ananalogue thereof and the CFS A [binding form (2)]; and a case when thebinding site to be bound by at least one kind of the CFS (for example,CFS A) among two or more of the CFSs is present only on the analyte orthe analogue thereof, and the binding site to be bound by another atleast one kind of the CFS (for example, CFS B) is present only on theCFS A [binding form (3)]. Among these, it is preferable that the bindingsites to be bound by each two or more of the CFSs are different. In thisconnection, in binding form (2), the substance having property tospecifically binding to a newly generated site (a CFS) includes, forexample, an antibody, a peptide chain and a nucleotide chain, and thelike, which can recognize a complex between an analyte or an analoguethereof and a CFS, and capable of binding thereto are included.

As a CFS as described above, one which binds with an analyte or ananalogue thereof by an “antigen”-“antibody” reaction or a “carbohydrate(sugar) chain”-“protein” reaction is preferable. Specifically, anantibody to an analyte or an analogue thereof, or an antigen bound withan analyte or an analogue thereof, or protein binding to an analyte oran analogue thereof is preferable, an antibody to an analyte or ananalogue thereof, or protein binding to an analyte or an analoguethereof is more preferable.

A CFS as described above may be bound with a labeling substance and/or asubstance capable of changing electrophoretic mobility (hereinafter,abbreviated as a reaction improvement substance) of an analyte or ananalogue thereof and may result in (1) a CFS having property capable offorming a complex with an analyte or an analogue thereof and capable ofchanging electrophoretic mobility of an analyte or an analogue thereof(hereinafter, abbreviated as a reaction improvement CFS or a reactionimprovement CFS), (2) a CFS having property capable of forming a complexwith an analyte or an analogue thereof and labeled with a labelingsubstance (a labeled CFS), and (3) a CFS labeled with a labelingsubstance, having property capable of forming a complex with an analyteor an analogue thereof and capable of changing electrophoretic mobilityof an analyte or an analogue thereof (a labeled reaction improvementCFS).

By using a CFS bound with a reaction improvement substance,electrophoretic mobility of a CFS can be changed, and arrangement orderof a solution containing an analyte or an analogue thereof, andsolutions including not less than one kind of CFSs in a step (1), canarbitrarily be controlled, and efficiency in concentration of an analyteor an analogue thereof and/or a CFS, and a reaction of an analyte or ananalogue thereof and a CFS (a complex formation reaction) can beenhanced.

In addition, by using a CFS bound with a labeling substance, an analytein a sample can be measured (detected).

(1) A Reaction Improvement CFS

A reaction improvement CFS is one having property capable of forming acomplex between an analyte or an analogue thereof and the substance, andcapable of changing electrophoretic mobility of an analyte or ananalogue thereof, in other words, one having property capable ofgenerating difference in behavior (electrophoretic mobility) of saidanalyte or analogue thereof corresponding to electrophoresis operationby forming a complex with an analyte or an analogue thereof, and onecapable of making electrophoretic mobility of a complex between ananalyte or an analogue thereof and a reaction improvement CFS (or acomplex between an analyte or an analogue thereof and a CFS other than areaction improvement CFS) higher or lower than electrophoretic mobility(faster or slower than electrophoretic mobility) of an analyte or ananalogue thereof itself (an analyte or an analogue thereof not boundwith a reaction improvement CFS).

As such a reaction improvement CFS, the above-described CFSs bound withthe following reaction improvement substances are general: For example,an inorganic metal oxide such as silica and alumina, etc.; a metal suchas gold, titanium, iron and nickel, etc.; an inorganic metal oxideintroduced with a functional group by an operation such as silanecoupling treatment, etc.; organisms such as various microorganisms andeukaryote cells, etc.; polysaccharide such as agarose, cellulose andinsoluble dextran, etc.; synthetic polymer compounds such as polystyrenelatex, a styrene-butadiene copolymer, a styrene-methacrylic acidcopolymer, an acrolein-ethyleneglycol dimethacrylate copolymer,styrene-styrenesulfonic acid latex, polyacrylamide, polyglycidylmethacrylate, polyacrolein coated particles, crosslinkedpolyacrylonitrile, acrylic acid or acrylate ester-based polymers, anacrylonitrile-butadiene copolymer, a vinyl chloride-acrylate estercopolymer and a poly vinyl acetate-acrylate copolymer, etc.;biomolecules such as erythrocyte, sugar, nucleic acid (polynucleotidesuch as RNA, DNA), protein, polypeptide and polyamino acid (polyglutamicacid, polyaspartic acid, polylysine, etc.); lipids; and the like.

However, for example, a reaction improvement substance may be bound toan analyte or an analogue thereof by a chemical binding method such as amethod for introducing a functional group at the surface of a reactionimprovement substance and subsequently binding to an analyte or ananalogue thereof via this functional group; by a method for bindingbetween a reaction improvement substance and an analyte or an analoguethereof via a linker; and the like. In this connection, in the abovedescription, a reaction improvement substance is one having propertycapable of providing property as a reaction improvement CFS as describedabove to said CFS, by binding to a CFS. Namely, a reaction improvementsubstance is one having property capable of changing electrophoreticmobility of an analyte or an analogue thereof, in other words, onehaving property capable of generating difference in behavior(electrophoretic mobility) of said analyte or analogue thereofcorresponding to electrophoresis operation, via a CFS, by forming acomplex between an analyte or an analogue thereof and a CFS (or acomplex among an analyte or an analogue thereof, a reaction improvementCFS and a CFS other than a reaction improvement CFS), and thus iscapable of making electrophoretic mobility of a complex between ananalyte or an analogue thereof and a reaction improvement CFS higher orlower than electrophoretic mobility (faster or slower thanelectrophoretic mobility) of an analyte or an analogue thereof itself ora complex between an analyte or analogue thereof not bound with areaction improvement CFS and a CFS.

Among those, as a reaction improvement CFS, those obtained by bindingnucleic acid (a nucleotide chain), protein, polypeptide or polyaminoacid to a CFS as described above are preferable, and those obtained bybinding nucleic acid (a nucleotide chain) or polyamino acid to a CFS aremore preferable. In addition, as a reaction improvement substance, thoseincluding an antibody as a CFS are preferable, specifically, thoseobtained by binding nucleic acid (a nucleotide chain), protein,polypeptide or polyamino acid as a reaction improvement substance to anantibody as a CFS are preferable, and among others those obtained bybinding nucleic acid (a nucleotide chain) or polyamino acid, as areaction improvement substance, to an antibody as a CFS are particularlypreferable.

To make binding of a reaction improvement substance to a CFS, namely, toprepare a reaction improvement CFS, any common method used in this fieldmay be applied, for example, a known labeling method itself generallyused in known EIA, RIA, FIA methods or a hybridization method themselves(for example, Ikagaku Jikken Koza (Experimental Manual in MedicalChemistry), vol. 8, edited by Yuichi Yamamura, 1^(st) Ed., NakayamaShoten Co., Ltd., 1971; Zusetu (Illustrative Description) FluorescentAntibodies, Akira Kawao, 1^(st) Ed., Softscience Co., Ltd., 1983; EnzymeImmunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, 3^(rd) Ed.,Igaku-Shoin Ltd., 1987; Molecular Cloning, A Laboratory Manual, 2nd Ed.,J. Sambroock, E. F. Fritsch, T. Maniatis, Cold Spring Harbor LaboratoryPress; Handbook of Fluorescent Probe and Research Chemicals, 7^(th) Ed.,Chapter 8, Molecular Probe Inc.; WO 2002/082083); or a common methodutilizing a reaction between avidin (or streptoavidin) and biotin.

In this connection, when a reaction improvement CFS is used as a CFS,combined use (parallel use) of a labeled CFS as described above ispreferable. However, when a reaction improvement CFS itself ismeasurable (detectable) by any of a method, or enables to be labeled bya labeling substance, combined use of a labeled CFS is not necessary.

In addition, when a labeled CFS and a reaction improvement CFS are usedin combination (are used parallely) as a CFS, and as long as a complexamong 3 components of an analyte or an analogue thereof, a labeled CFSand a reaction improvement CFS, is formed, binding forms of these 3components or binding sites of a labeled CFS and a reaction improvementCFS are not especially limited. Such binding forms include, for example,(1) so-called a sandwich complex wherein an analyte or an analoguethereof is sandwiched by a labeled CFS and a reaction improvement CFS,(2) a complex wherein a reaction improvement CFS or a labeled CFS isfurther bound at a binding site with an analyte or an analogue thereofand a labeled CFS or a reaction improvement CFS and (3) a complexwherein a reaction improvement CFS or a labeled CFS is further bound ata labeled CFS or a reaction improvement CFS bound with an analyte or ananalogue thereof, and the like. In addition, said binding moietiesinclude, for example, (1) the case when all of the binding sites of alabeled CFS and a reaction improvement CFS are present only on ananalyte or an analogue thereof [binding form (1)], (2) the case wheneither of the binding sites of a labeled CFS or a reaction improvementCFS is present only on an analyte and an analogue thereof, and the otherbinding site is present at a new site generated by formation of acomplex between an analyte and either of said labeled CFS and reactionimprovement CFS [binding form (2)], (3) either of binding sites of alabeled CFS and a reaction improvement CFS is present only on an analyteor an analogue thereof, while the other binding site is present at onlyeither of said labeled CFS and reaction improvement CFS [binding form(3)] and (4) the case of combinations thereof. Among those, a bindingsite of a labeled CFS, and a binding site of a reaction improvement CFSis preferably a different one. In this connection, in the abovedescription (2), as one having property to specifically bind at a newlygenerated site (a labeled CFS and/or a reaction improvement CFS), forexample, an antibody, a peptide chain, a nucleotide chain, and the like,which enable to recognize a complex between an analyte or an analoguethereof and a labeled CFS and/or a reaction improvement CFS, andbindable thereto are included.

(2) A Labeled CFS

A CFS as described above is generally one measurable (detectable) itselfby any of a method, or enables to be labeled by a labeling substance. Byusing one having such property, an analyte in a sample can be measured(detected). In this connection, when an analyte or an analogue thereofitself is detectable by any of a method (for example, an enzyme, and thelike), or an analyte or an analogue thereof is directly bindable to alabeling substance without using (not through) a CFS, an analyte in asample can be measured (detected) even when said CFS may not have theabove-described property. Examples of those detectable themselves by anyof a method include an enzyme, a dye, a fluorescent substance, aluminescent substance, a substance having absorption at UV region, andthe like.

Among those, as a CFS used in the present invention, a substanceformable a complex with an analyte or an analogue thereof, and labeledby a labeling substance (a labeled CFS) is preferable.

As a labeling substance used in the present invention, any one used inthis field such as an enzyme immunoassay (EIA), a radio immunoassay(RIA), a fluorescent immunoassay (FIA), a hybridization method, and thelike, may be adopted. Such a labeling substance includes, for example,enzymes such as alkaline phosphatase (ALP), β-galactosidase (β-Gal),peroxidase (POD), microperoxidase, glucose oxidase (GOD),glucose-6-phophate dehydrogenase (G6PDH), malate dehydrogenase andluciferase, etc.; dyes such as Coomassie brilliant blue R250, and methylorange, etc.; radioactive isotopes such as ^(99m)Tc, ¹³¹I, ¹²⁵I, ¹⁴C,³H, ³²P and ³⁵S, etc.; HiLyte type dyes such as HiLyte Fluor 488, HiLyteFluor 555, HiLyte Fluor 647, HiLyte Fluor 680 and HiLyte Fluor 750, etc.(all of them are trade names of HiLyte Bioscience, Inc.); Alexa typedyes such as Alexa Fluor Dye 350, Alexa Fluor Dye 430, Alexa Fluor Dye488, Alexa Fluor Dye 532, Alexa Fluor Dye 546, Alexa Fluor Dye 555,Alexa Fluor Dye 568, Alexa Fluor Dye 594, Alexa Fluor Dye 633, AlexaFluor Dye 647, Alexa Fluor Dye 660, Alexa Fluor Dye 680, Alexa Fluor Dye700 and Alexa Fluor Dye 750 etc. (all of them are trade names ofMolecular Probes, Inc.); CyDye type dyes such as Cy3, Cy3.5, Cy5, Cy5.5and Cy7, etc. (all of them are trade names of Amersham Biosciences,Inc.); fluorescent materials such as fluorescein, rhodamine, dansyl,fluorescamine, coumarin, naphthylamine, or derivatives thereof,rare-earth fluorescent dyes [combinations of a rare earth metal such assamarium (Sm), europium (Eu), terbium (Tb) or dysprosium (Dy) and achelate compound such as4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedione-6″-yl)chlorosulfo-O-terphenyl (BHHCT),4,7-bis(chlorosulfonyl)-1,10-phenanthoroline-2,9-dicarboxylic acid(BCPDA), β-naphthyltrifluoroacetic acid (β-NTA), etc.], intercalatordyes [for example, acridine dyes such as acridine orange, etc.; ethidiumcompounds such as ethidium bromide, ethidium homodimer-1 (EthD-1),ethidium homodimer-2 (EthD-2), ethidium bromide monoazide (EMA) anddihydroethidium, etc.; iodine compounds such as propidium iodide, andhexidium iodide, etc.; 7-aminoactinomycin D (7-AAD); cyanine dimmer typedyes such as POPO-1, BOBO-1, YOYO-1, TOTO-1, JOJO-1, POPO-3, LOLO-1,BOBO-3, YOYO-3, and TOTO-3, etc. (all of them are trade names ofMolecular Probes, Inc.); SYTOX type dyes such as SYBR Gold, SYBR Green Iand SYBR Green II, SYTOX Green, SYTOX Blue, and SYTOX Orange, etc. (allof them are trade names of Molecular Probes, Inc.), and the like], onebound to a minor group of DNA double helix [for example,4′,6-diamidino-2-phenylindole (DAPI: trade name of Molecular Probes,Inc.), pentahydrate(bis-benzimido) (Hoechst 33258: trade name ofMolecular Probes, Inc.), and trihydrochloride (Hoechst 33342: trade nameof Molecular Probes, Inc.), etc.]; benzimido type dyes (Hoechst 34580:trade name of Molecular Probes, Inc.) and the like], one specificallybound to the sequence of adenine-thymine (A-T) [for example, acridinedyes such as 9-amino-6-chloro-2-methoxyacridine (ACMA), andbis(6-chloro-2-methoxy-9-acridinyl) spermine (acridine homodimer), etc.,hydroxystilbamidine, and the like]; luminescent materials such asluciferin, isoluminal, luminal, and bis(2,4,6-trifluorophenyl) oxalate,etc.; material having absortion in ultra-violet region such as phenol,naphthol, anthracene, or derivatives thereof; substances having propertyas spin labeling agents represented by compounds having an oxyl groupsuch as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl,3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, and2,6-di-t-butyl-α-(3,5-di-t-butyl-4-oxo-2,5-cyclohexadine-1-ylidene)-p-tolyloxyl;and the like.

Labeling a CFS with a labeling substance may be carried out according toa method similar to a method for labeling a CFS with a labelingsubstance as described above, or a common method described in WO2002/082083.

(3) A Labeled Reaction Improvement CFS

Furthermore, in the present invention, as a CFS, such a substance mayalso be used that is labeled by a labeling substance, formable a complexwith an analyte or an analogue thereof, and capable of changingelectrophoretic mobility of an analyte (a labeled reaction improvementCFS), namely, a reaction improvement CFS labeled by a labelingsubstance. In this connection, a labeling substance and a reactionimprovement CFS are as described above, and also labeling a reactionimprovement CFS with a labeling substance may be carried out accordingto a method similar to a method for labeling a CFS with a labelingsubstance as described above, or a common method described in WO2002/082083.

(4) Combinations of CFSs

As described above, in the present invention, for example, the followingvarious CFSs are used. In this connection, they may naturally be used insuitable combinations.

(a) A CFS not bound with a labeling substance and a reaction improvementsubstance

(b) A labeled CFS

(c) A reaction improvement CFS

(d) A labeled reaction improvement CFS

(e) A CFS not bound with a labeling substance and a reaction improvementsubstance and a reaction improvement CFS

(f) A labeled CFS and a reaction improvement CFS

(g) A CFS not bound with a labeling substance, and a reactionimprovement substance, and a labeled reaction improvement CFS

(5) A Solution Containing a CFS

As a solution containing a CFS of the present invention, as describedabove, any one may be used as long as it does not inhibit formation of acomplex between an analyte or an analogue thereof and said CFS. Suchsolution includes, for example, water, a buffer solution, and the like.

As such a buffer solution, any one may be used as long as it has bufferaction usually in a pH range of 5 to 11, and does not inhibit formationof said complex formation reaction. Examples of the buffer solution arethose usually used in this field such as Tris buffer, Good's buffer, TEbuffer, TAE buffer, TBE buffer, TBS buffer, a phosphate buffer, a boratebuffer, and the like. Use concentration of these buffers is usually 1 mMto 2 M, preferably 10 mM to 1 M, and pH is usually 5 to 11, preferably 5to 10, more preferably 5.5 to 8.5, further preferably 6 to 8 andparticularly preferably around 7.

Concentration of a CFS contained in a solution as described above,namely use amount of a CFS is not simply described due to dependency onkinds of CFSs used, however, it is usually preferable that the CFS ispresent in the reaction solution (a solution containing an analyte or ananalogue thereof and a CFS) at a concentration which is not lower than(preferably not lower than 2 times, more preferably not lower than 5times) a concentration at which the CFS can bind to the whole of theanalytes or analogues thereof of a concentration corresponding to thelimit of measurement.

In addition, upper limit of the concentration is not especially limited,however, in view of economical efficiency, it is usually not higher than10¹² times (preferably not higher than 10⁹ times, more preferably nothigher than 10⁶ times) a concentration at which the CFS can bind to thewhole of the analytes or analogues thereof of a concentrationcorresponding to the limit of measurement.

In more specifically, a CFS may be contained in a solution as describedabove, so that concentration of a CFS in a solution containing ananalyte or an analogue thereof and a CFS is, as lower limit, usually notlower than 10 pM, preferably not lower than 1 nM, and more preferablynot lower than 100 nM, and as upper limit, usually not higher than 10μM, preferably not higher than 1 μM and more preferably not higher than500 nM.

1-6. Specific Methods for Forming a Complex

Modes for carrying out the methods for forming a complex of the presentinvention are specifically shown below.

(a) A Case when a CFS not Bound with a Labeling Substance and a ReactionImprovement Substance are Used.

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs] and (b) a solutioncontaining not less than one kind of CFSs are introduced and arrangedinto a capillary so that a zone of the solution containing an analyte[for example, (i) a sample including an analyte, (ii) a solutionincluding a sample having an analyte, and not less than one kind ofCFSs], and a zone of the solution including not less than one kind ofCFSs are separately formed (so that liquid-liquid interface is formed),and a complex between said analyte and CFS is formed on application of avoltage onto said capillary, without mixing these solutions in advanceoutside a capillary.

(2) Subsequently, said analyte is electrophoretically contacted withsaid CFS while concentrating said analyte and/or at least one kind ofthe CFSs by applying a voltage to said capillary before uniformly mixingthese solutions, not by (not depending on) molecular diffusion andwithout physically mixing, to form the complex between said analyte andthe CFS.

In the above description, “before these solutions are uniformly mixed”means “before each zone (liquid-liquid interface) of a solutioncontaining an analyte [for example, (i) a sample including an analyte,(ii) a solution including a sample having an analyte, and not less thanone kind of CFSs], and a solution containing not less than one kind ofCFSs, along with if necessary the liquid, arranged in a capillary by astep (1) are uniformly mixed by molecular diffusion”. In thisconnection, “interface” means the same as described above.

In addition, in the above-described step (2), “concentrating saidanalyte and/or at least one kind of CFSs by applying a voltage onto acapillary” means that, as similarly described above, said analyte and/orat least one kind of CFS gather in band-like (plug-like) on applicationof a voltage onto a capillary. In other words, it means that saidsubstances gather on application of a voltage onto a capillary so thatsuch a portion is generated wherein concentration of said substancesbecomes higher than that of a substance in a zone arranged in a step(1), namely it means that an analyte and/or at least one kind of a CFSgarher on application of a voltage onto a capillary, and a portion isgenerated wherein concentration of an analyte and/or concentration ofnot less than one kind of CFSs becomes higher than that of an analyteand/or not less than one kind of CFSs in a solution zone [for example, azone of a solution containing an analyte (for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs) and a zone of a solutioncontaining not less than one kind of CFSs] arranged in a step (1).

In the above step (2), “said analyte is electrophoretically contactedwith said CFS” means that, similarly as described above, contact of saidanalyte and CFS is conducted, not by (not depending on) moleculardiffusion and by utilization of the phenomenon that when a solutioncontaining a substance with higher electrophoretic mobility (fasterelectrophoretic speed) is arranged at upstream of a solution containinga substance with lower electrophoretic mobility (slow electrophoreticspeed), and the electrophoresis is conducted, a substance with higherelectrophoretic mobility (faster electrophoretic speed) in a solutionovertakes a substance with lower electrophoretic mobility (slowelectrophoretic speed).

(b) A Case when a Labeled CFS is Used.

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs] and (b) a solutioncontaining not less than one kind of labeled CFSs are introduced andarranged into a capillary so that a zone of the solution containing ananalyte [for example, (i) a sample including an analyte, (ii) solutionsincluding a sample having an analyte, and not less than one kind ofCFSs], and a zone of the solution containing not less than one kind oflabeled CFSs are separately formed (so that liquid-liquid interface isformed), and a complex between said analyte and labeled CFS is formed onapplication of a voltage onto a capillary, without mixing thesesolutions in advance outside a capillary.

(2) Subsequently, said analyte is electrophoretically contacted withsaid labeled CFS while concentrating said analyte and/or at least onekind of said labeled CFS by applying a voltage onto said capillarybefore uniformly mixing these solutions, not by (not depending on)molecular diffusion and without physically mixing, to form the complexbetween said analyte and labeled CFS.

In the above description, “before these solutions are uniformly mixed”means “before each zone (liquid-liquid interface) of a solutioncontaining an analyte [for example, (i) a sample including an analyte,(ii) solutions including a sample having an analyte, and not less thanone kind of CFSs], and a solution containing not less than one kind oflabeled CFSs, along with if necessary the liquid, arranged in acapillary by a step (1) are uniformly mixed by molecular diffusion”. Inthis connection, “interface” means the same as described above.

In addition, in the above-described step (2), “concentrating saidanalyte and/or at least one kind of labeled CFSs by applying a voltageonto a capillary” means that, as similarly described above, said analyteand/or at least one kind of labeled CFS to gather in band-like(plug-like) on application of a voltage onto a capillary. In otherwords, it means that said substances gather on application of a voltageonto a capillary so that such a portion is generated whereinconcentration of said substances becomes higher than that of a substancein a zone arranged in a step (1), namely, it means that an analyteand/or at least one kind of a labeled CFS garher on application of avoltage onto a capillary, and a portion is generated whereinconcentration of an analyte and/or concentration of at least one kind ofa labeled CFS becomes higher than that of an analyte and/or at least onekind of a labeled CFS in a solution zone [for example, a zone of asolution containing an analyte (for example, (i) a sample including ananalyte, (ii) a solution including a sample having an analyte, and notless than one kind of CFSs) and a zone of a solution containing not lessthan one kind of labeled CFSs] arranged in a step (1).

In the above step (2), “said analyte is electrophoretically contactedwith said labeled CFS” means that, similarly as described above, contactof said analyte and labeled CFS is conducted, not by (not depending on)molecular diffusion and by utilization of the phenomenon that a solutioncontaining a substance with higher electrophoretic mobility (fasterelectrophoretic speed) is arranged at upstream of a solution containinga substance with lower electrophoretic mobility (slow electrophoreticspeed), and the electrophoresis is conducted, a substance with higherelectrophoretic mobility (faster electrophoretic speed) in a solutionovertakes a substance with lower electrophoretic mobility (slowelectrophoretic speed).

(c) A Case when a Reaction Improvement CFS is Used.

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs] and (b) a solutioncontaining not less than one kind of reaction improvement CFSs areintroduced and arranged into a capillary so that a zone of the solutioncontaining an analyte [for example, (i) a sample including an analyte,(ii) a solution including a sample having an analyte, and not less thanone kind of CFSs], and a zone of the solution including not less thanone kind of reaction improvement CFSs are separately formed (so thatliquid-liquid interface is formed), and a complex between said analyteand reaction improvement CFS is formed on application of a voltage ontoa capillary, without mixing these solutions in advance outside acapillary.

(2) Subsequently, said analyte is electrophoretically contacted withsaid reaction improvement CFS while concentrating said analyte and/or atleast one kind of the reaction improvement CFS by applying a voltageonto said capillary before uniformly mixing these solutions, not by (notdepending on) molecular diffusion and without physically mixing, to formthe complex between said analyte and reaction improvement CFS.

In the above description, “before these solutions are uniformly mixed”means “before each zone (liquid-liquid interface) of a solutioncontaining an analyte [for example, (i) a sample including an analyte,(ii) a solution including a sample having an analyte, and not less thanone kind of CFSs], and a solution containing not less than one kind ofreaction improvement CFSs, along with if necessary the liquid, arrangedin a capillary by a step (1) are uniformly mixed by moleculardiffusion”. In this connection, “interface” means the same as describedabove.

In addition, in the above-described step (2), “concentrating saidanalyte and/or at least one kind of reaction improvement CFSs byapplying a voltage onto a capillary” means that, as similarly describedabove, said analyte and/or at least one kind of reaction improvement CFSgather in band-like (plug-like) on application of a voltage onto acapillary. In other words, it means that said substances gather onapplication of a voltage onto a capillary so that such a portion isgenerated wherein concentration of said substances becomes higher thanthat of a substance in a zone arranged in a step (1), namely, it meansthat an analyte and/or at least one kind of a reaction improvement CFSgarher on application of a voltage onto a capillary, and a portion isgenerated wherein concentration of an analyte and/or concentration ofnot less than one kind of reaction improvement CFSs becomes higher thanthat of an analyte and/or not less than one kind of reaction improvementCFSs in a solution zone [for example, a solution zone containing ananalyte (for example, (i) a sample including an analyte, (ii) a solutionincluding a sample having an analyte, and not less than one kind ofCFSs) and a zone of a solution containing not less than one kind ofreaction improvement CFSs] arranged in a step (1).

In the above step (2), “said analyte is electrophoretically contactedwith said reaction improvement substance?)” means that, similarly asdescribed above, contact of said analyte and reaction improvement CFS isconducted, not by (not depending on) molecular diffusion and byutilization of the phenomenon that when a solution containing asubstance with higher electrophoretic mobility (faster electrophoreticspeed) is arranged at upstream of a solution containing a substance withlower electrophoretic mobility (slow electrophoretic speed), theelectrophoresis is conducted, a substance with higher electrophoreticmobility (faster electrophoretic speed) in a solution overtakes asubstance with lower electrophoretic mobility (slow electrophoreticspeed).

(d) A Case when a Labeled Reaction Improvement CFS is Used.

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs] and (b) a solutioncontaining not less than one kind of labeled reaction improvement CFSsare introduced and arranged into a capillary so that a zone of thesolution containing an analyte [for example, (i) a sample including ananalyte, (ii) a solution including a sample having an analyte, and notless than one kind of CFSs], and a zone of a solution including not lessthan one kind labeled reaction improvement CFSs are separately formed(so that liquid-liquid interface is formed), and a complex between saidanalyte and labeled reaction improvement CFS is formed on application ofa voltage onto said capillary, without mixing these solutions in advanceoutside a capillary.

(2) Subsequently, said analyte is electrophoretically contacted saidlabeled reaction improvement CFS while concentrating said analyte and/orat least one kind of a labeled reaction improvement CFS by applying avoltage onto said capillary before uniformly mixing these solutions, notby (not depending on) molecular diffusion and without physically mixing,to form the complex between said analyte and labeled reactionimprovement CFS.

In the above description, “before these solutions are uniformly mixed”means “before each zone (liquid-liquid interface) of a solutioncontaining an analyte [for example, (i) a sample including an analyte,(ii) a solution including a sample having an analyte, and not less thanone kind of CFSs], and a solution including not less than one kindlabeled reaction improvement CFSs, along with if necessary the liquid,arranged in a capillary by a step (1) are uniformly mixed by moleculardiffusion”. In this connection, “interface” means the same as describedabove.

In addition, in the above-described step (2), “concentrating saidanalyte and/or at least one kind of labeled reaction improvement CFS byapplying a voltage onto a capillary” means that, as similarly describedabove, for said analyte and/or at least one kind of labeled reactionimprovement CFS gather in band-like (plug-like) on application of avoltage onto a capillary. In other words, it means that said substancesgather on application of a voltage onto a capillary so that such aportion is generated wherein concentration of said substances becomeshigher than a substance in a zone arranged in a step (1), namely, itmeans that an analyte and/or at least one kind of a labeled reactionimprovement CFS garher on application of a voltage onto a capillary, anda portion is generated wherein concentration of an analyte and/orconcentration of not less than one kind of labeled reaction improvementCFSs becomes higher than that of an analyte and/or not less than onekind of labeled reaction improvement CFSs in a solution zone [forexample, a zone of a solution containing an analyte (for example, (i) asample including an analyte, (ii) a solution including a sample havingan analyte, and not less than one kind of CFSs) and a zone of a solutioncontaining not less than one kind of labeled reaction improvement CFSs]arranged in a step (1).

In the above step (2), “said analyte is electrophoretically contactedwith said labeled reaction improvement CFS?)” means that, similarly asdescribed above, contact of said analyte and labeled reactionimprovement CFS is conducted, not by (not depending on) moleculardiffusion and by utilization of the phenomenon that when a solutioncontaining a substance with higher electrophoretic mobility (fasterelectrophoretic speed) is arranged at upstream of a solution containinga substance with lower electrophoretic mobility (slow electrophoreticspeed), and the electrophoresis is conducted, a substance with higherelectrophoretic mobility (faster electrophoretic speed) in a solutionovertakes a substance with lower electrophoretic mobility (slowelectrophoretic speed).

(e) A Case when a CFS not Bound with a Labeling Substance and a ReactionImprovement Substance, and a Reaction Improvement CFS are Used.

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs], (b) a solution containingnot less than one kind of a CFS and (c) a solution containing not lessthan one kind of a reaction improvement CFS are introduced and arrangedinto a capillary so that a zone of the solution containing an analyte[for example, (i) a sample including an analyte, (ii) a solutionincluding a sample having an analyte, and not less than one kind ofCFSs], a zone of the solution including not less than one kind of CFSs,and a zone of the solution including not less than one kind of reactionimprovement CFSs are separately formed (so that liquid-liquid interfaceis formed), and a complex between said analyte, CFS and reactionimprovement CFS is formed on application of a voltage onto a capillary,without mixing these solutions in advance outside a capillary.

(2) Subsequently, said analyte is electrophoretically contacted withsaid CFS and reaction improvement CFS while concentrating at least oneselected from said analyte, not less than one kind of CFS and not lessthan one kind of reaction improvement CFS by applying a voltage ontosaid capillary before uniformly mixing these solutions, not by (notdepending on) molecular diffusion and without physically mixing, to formthe complex between said analyte, CFS and reaction improvement CFS.

In the above description, “before these solutions are uniformly mixed”means “before each zone (liquid-liquid interface) of a solutioncontaining an analyte [for example, (i) a sample including an analyte,(ii) a solution including a sample having an analyte, and not less thanone kind of CFSs], a solution including not less than one kind of CFSs,and a solution including not less than one kind of reaction improvementCFSs, along with if necessary the liquid, arranged in a capillary by astep (1) are uniformly mixed by molecular diffusion”. In thisconnection, “interface” means the same as described above.

In addition, in the above-described step (2), “concentrating at leastone selected from said analyte not less than one kind of CFS and notless than one kind of reaction improvement CFS, by applying a voltageonto a capillary” means that, as similarly described above, at least oneselected from said analyte, not less than one kind of CFS and not lessthan one kind of reaction improvement CFS gather in band-like(plug-like) on application of a voltage onto a capillary. In otherwords, it means that said substances gather on application of a voltageonto a capillary so that such a portion is generated whereinconcentration of said substances becomes higher than that of a substancein a zone arranged in a step (1), namely, it means that at least oneselected from an analyte, not less than one kind of CFS and not lessthan one kind of reaction improvement CFS garher on application of avoltage onto a capillary, and a portion is generated whereinconcentration of an analyte, concentration of not less than one kind ofa CFS or concentration of not less than one kind of a reactionimprovement CFS becomes higher than that of an analyte, not less thanone kind of a CFS or not less than one kind of a reaction improvementCFS in a solution zone [for example, a zone of a solution containing ananalyte (for example, (i) a sample including an analyte, (ii) a solutionincluding a sample having an analyte, and not less than one kind ofCFSs), a zone of a solution including not less than one kind of CFSs anda zone of a solution including not less than one kind of reactionimprovement CFSs] arranged in a step (1).

In the above step (2), “said analyte is electrophoretically contactedwith said CFS and reaction improvement CFS” means_that, similarly asdescribed above, contact of said analyte, CFS and reaction improvementCFS is conducted, not by (not depending on) molecular diffusion and byutilization of the phenomenon that when a solution containing asubstance with higher electrophoretic mobility (faster electrophoreticspeed) is arranged at upstream of a solution containing a substance withlower electrophoretic mobility (slow electrophoretic speed), and theelectrophoresis is conducted, a substance with higher electrophoreticmobility (faster electrophoretic speed) in a solution overtakes asubstance with lower electrophoretic mobility (slow electrophoreticspeed).

(f) A Case when a Labeled CFS and a Reaction Improvement CFS are Used.

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs], (b) a solution containingnot less than one kind of a labeled CFS and (c) a solution containingnot less than one kind of a reaction improvement CFS are introduced andarranged into a capillary so that a zone of the solution containing ananalyte [for example, (i) a sample including an analyte, (ii) a solutionincluding a sample having an analyte, and not less than one kind ofCFSs], a zone of the solution containing not less than one kind oflabeled CFSs, and a zone of the solution containing not less than onekind of reaction improvement CFSs are separately formed (so thatliquid-liquid interface is formed), and a complex between said analyte,labeled CFS and reaction improvement CFS is formed on application of avoltage onto a capillary, without mixing these solutions in advanceoutside a capillary.

(2) Subsequently, said analyte is electrophoretically contacted withsaid labeled CFS and reaction improvement CFS while concentrating atleast one selected from said analyte, not less than one kind of labeledCFS and not less than one kind of reaction improvement CFS by applying avoltage onto said capillary before uniformly mixing these solutions, notby (not depending on) molecular diffusion and without physically mixing,to form the complex between said analyte, labeled CFS and reactionimprovement CFS.

In the above description, “before these solutions are uniformly mixed”means “before each zone (liquid-liquid interface) of a solutioncontaining an analyte [for example, (i) a sample including an analyte,(ii) a solution including a sample having an analyte, and not less thanone kind of CFSs], a solution including not less than one kind oflabeled CFSs, and a solution including not less than one kind ofreaction improvement CFSs along with if necessary the liquid, arrangedin a capillary by a step (1) are uniformly mixed by moleculardiffusion”. In this connection, “interface” means the same as describedabove.

In addition, in the above-described step (2), “concentrating at leastone selected from said analyte, not less than one kind of labeled CFSand not less than one kind of reaction improvement CFS, by applying avoltage onto a capillary” means that, as similarly described above, atleast one selected from said analyte, not less than one kind of labeledCFS and not less than one kind of reaction improvement CFS gather inband-like (plug-like) on application of a voltage onto a capillary. Inother words, it means that said substances gather on application of avoltage onto a capillary so that such a portion is generated whereinconcentration of said substances becomes higher than a substance in azone arranged in a step (1), namely, it means that at least one selectedfrom an analyte, not less than one kind of labeled CFS and not less thanone kind of reaction improvement CFS garher on application of a voltageonto a capillary, and a portion is generated wherein concentration of ananalyte, concentration of not less than one kind of a labeled CFS orconcentration of not less than one kind of a reaction improvement CFSbecomes higher than that of an analyte, not less than one kind of alabeled CFS or not less than one kind of a reaction improvement CFS in asolution zone [for example, a zone of a solution containing an analyte(for example, (i) a sample including an analyte, (ii) a solutionincluding a sample having an analyte, and not less than one kind ofCFSs), a zone of a solution including not less than one kind of alabeled CFS, and a zone of a solution including not less than one kindof a reaction improvement CFS] arranged in a step (1).

In the above step (2), “said analyte is electrophoretically contactedwith said labeled CFS and reaction improvement CFS” means_that,similarly as described above, contact of said analyte, labeled CFS andreaction improvement CFS is conducted, not by (not depending on)molecular diffusion and by utilization of the phenomenon that when asolution containing a substance with higher electrophoretic mobility(faster electrophoretic speed) is arranged at upstream of a solutioncontaining a substance with lower electrophoretic mobility (slowelectrophoretic speed), and the electrophoresis is conducted, asubstance with higher electrophoretic mobility (faster electrophoreticspeed) in a solution overtakes a substance with lower electrophoreticmobility (slow electrophoretic speed).

(g) A Case when a CFS not Bound with a Labeling Substance and a ReactionImprovement Substance, and a Labeled Reaction Improvement CFS are Used.

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs], (b) a solution containingnot less than one kind of a CFS and (c) a solution containing not lessthan one kind of a labeled reaction improvement CFS are introduced andarranged into a capillary so that a zone of a solution containing ananalyte [for example, (i) a sample including an analyte, (ii) a solutionincluding a sample having an analyte, and not less than one kind ofCFSs], a zone of the solution including not less than one kind of CFSs,and a zone of the solution containing not less than one kind labeledreaction improvement CFSs are separately formed (so that liquid-liquidinterface is formed), and a complex between said analyte, CFS andlabeled reaction improvement CFS is formed on application of a voltageonto a capillary, without mixing these solutions in advance outside acapillary.

(2) Subsequently, said analyte is electrophoretically contacted withsaid CFS and labeled reaction improvement CFS while concentrating atleast one selected from said analyte, not less than one kind of CFS andnot less than one kind of labeled reaction improvement CFS by applying avoltage onto said capillary before uniformly mixing these solutions, notby (not depending on) molecular diffusion and without physically mixing,to form the complex between said analyte, CFS and labeled reactionimprovement CFS.

In the above description, “before these solutions are uniformly mixed”means “before each zone (liquid-liquid interface) of a solutioncontaining an analyte [for example, (i) a sample including an analyte,(ii) a solution including a sample having an analyte, and not less thanone kind of CFSs], a solution including not less than one kind of CFSs,and a solution containing not less than one kind labeled reactionimprovement CFSs, along with if necessary the liquid, arranged in acapillary by a step (1) are uniformly mixed by molecular diffusion”. Inthis connection, “interface” means the same as described above.

In addition, in the above-described step (2), “concentrating at leastone selected from said analyte, not less than one kind of CFS and notless than one kind of labeled reaction improvement CFS, by applying avoltage onto a capillary” means that, as similarly described above, atleast one selected from said analyte, not less than one kind of CFS andnot less than one kind of labeled reaction improvement CFS gather inband-like (plug-like) on application of a voltage onto a capillary. Inother words, it means that said substances gather on application of avoltage onto a capillary so that such a portion is generated whereinconcentration of said substances becomes higher than a substance in azone arranged in a step (1), namely, it means that at least one selectedfrom an analyte, not less than one kind of CFS and not less than onekind of labeled reaction improvement CFS garher on application of avoltage onto a capillary, and a portion is generated whereinconcentration of an analyte, concentration of not less than one kind ofa CFS or concentration of not less than one kind of a labeled reactionimprovement CFS becomes higher than that of an analyte, not less thanone kind of a CFS or not less than one kind of a labeled reactionimprovement CFS in a solution zone [for example, a zone of solutioncontaining an analyte (for example, (i) a sample including an analyte,(ii) a solution including a sample having an analyte, and not less thanone kind of CFSs), a zone of a solution including not less than one kindof CFSs and a zone of a solution containing not less than one kindlabeled reaction improvement CFSs] arranged in a step (1).

In the above step (2), “said analyte is electrophoretically contactedwith said CFS and labeled reaction improvement CFS” means that,similarly as described above, contact of said analyte, CFS and labeledreaction improvement CFS is conducted, not by (not depending on)molecular diffusion and by utilization of the phenomenon that when asolution containing a substance with higher electrophoretic mobility(faster electrophoretic speed) is arranged at upstream of a solutioncontaining a substance with lower electrophoretic mobility (slowelectrophoretic speed), and the electrophoresis is conducted, asubstance with higher electrophoretic mobility (faster electrophoreticspeed) in a solution overtakes a substance with lower electrophoreticmobility (slow electrophoretic speed).

A method for forming a complex of the present invention can also be usedin so-called a competitive method. The procedure in carrying it out in acompetitive method is as follows:

(h) A Case when a Labeled Analogue and a CFS are Used.

(1) (a) A solution containing a sample having an analyte and a labeledanalogue (or a solution including a sample having an analyte, a labeledanalogue and not less than one kind of CFSs) and (b) a solutioncontaining not less than one kind of CFSs are introduced and arrangedinto a capillary so that a zone of the solution containing a samplehaving an analyte and a labeled analogue (or the solution including asample having an analyte, a labeled analogue and not less than one kindof CFSs), and a zone of the solution including not less than one kind ofCFSs are separately formed (so that liquid-liquid interface is formed),and a complex A between said analyte and CFS and a complex B betweensaid labeled analogue and CFS are formed on application of a voltageonto a capillary, without mixing these solutions in advance outside acapillary.

(2) Subsequently, said analyte and labeled analogue areelectrophoretically contacted with said CFS (namely, said analyte iselectrophoretically contacted with said CFS, and said labeled analogueelectrophoretically contacted with said CFS) while concentrating saidanalyte and labeled analogue and/or not less than one kind of a CFS byapplying a voltage onto said capillary before uniformly mixing thesesolutions, not by (not depending on) molecular diffusion and withoutphysically mixing, to form the complex A between said analyte and CFSand a complex B between said labeled analogue and CFS.

In the above description, “before these solutions are uniformly mixed”means “before each zone (liquid-liquid interface) of a solutionincluding a sample having an analyte and a labeled analogue (or asolution including a sample having an analyte, a labeled analogue andnot less than one kind of CFSs), and a solution including not less thanone kind of CFSs, along with if necessary the liquid, arranged in acapillary by a step (1) are uniformly mixed by molecular diffusion”. Inthis connection, “interface” means the same as described above.

In addition, in the above-described step (2), “concentrating saidanalyte and labeled analogue, and/or not less than one kind of a CFS, byapplying a voltage onto a capillary” means that, as similarly describedabove, said analyte and labeled analogue and/or not less than one kindof CFS gather in band-like (plug-like) on application of a voltage ontoa capillary. In other words, it means that said substances gather onapplication of a voltage onto a capillary so that such a portion isgenerated wherein concentration of said substances becomes higher than asubstance in a zone arranged in a step (1), namely, it means that ananalyte and a labeled analogue, and/or not less than one kind of a CFSgarher on application of a voltage onto a capillary, and a portion isgenerated wherein concentration of an analyte and a labeled analogue,and/or concentration of a CFS becomes higher than that of an analyte anda labeled analogue, and/or not less than one kind of a CFS in a solutionzone [for example, a zone of a solution including a sample having ananalyte and a labeled analogue (or a solution including a sample havingan analyte, a labeled analogue and not less than one kind of CFSs), azone of a solution containing CFSs] arranged in a step (1).

In the above step (2), “said analyte and labeled analogue areelectrophoretically contacted with said CFS (namely, said analyte iselectrophoretically contacted with said CFS, and said labeled analogueelectrophoretically contacted with said CFS)” means that, similarly asdescribed above, contact of said analyte, labeled analogue and CFS areconducted (contact of each of said analyte and CFS, and labeled analogueand CFS are conducted), not by (not depending on) molecular diffusionand by utilization of the phenomenon that when a solution containing asubstance with higher electrophoretic mobility (faster electrophoreticspeed) is arranged at upstream of a solution containing a substance withlower electrophoretic mobility (slow electrophoretic speed), and theelectrophoresis is conducted, a substance with higher electrophoreticmobility (faster electrophoretic speed) in a solution overtake asubstance with lower electrophoretic mobility (slow electrophoreticspeed).

(i) A Case when a Labeled Analogue and a Reaction Improvement CFS areUsed.

(1) (a) A solution including a sample having an analyte and a labeledanalogue (or a solution including a sample having an analyte, a labeledanalogue and not less than one kind of CFSs) and (b) a solutioncontaining not less than one kind of reaction improvement CFSs areintroduced and arranged into a capillary so that a zone of the solutionincluding a sample having an analyte and a labeled analogue (or thesolution including a sample having an analyte, a labeled analogue andnot less than one kind of CFSs), and a zone of the solution includingnot less than one kind of reaction improvement CFSs are separatelyformed (so that liquid-liquid interface is formed), and a complex Abetween said analyte and reaction improvement CFS and a complex Bbetween said labeled analogue and reaction improvement CFS are formed onapplication of a voltage onto a capillary, without mixing thesesolutions in advance outside a capillary.

(2) Subsequently, said analyte and labeled analogue areelectrophoretically contacted with said reaction improvement CFS(namely, said analyte is electrophoretically contacted with saidreaction improvement CFS, and said labeled analogue iselectrophoretically contacted with said reaction improvement CFS) whileconcentrating said analyte and labeled analogue and/or not less than onekind of said reaction improvement CFS by applying a voltage onto saidcapillary before uniformly mixing these solutions, not by (not dependingon) molecular diffusion and without physically mixing, to form thecomplex A between said analyte and reaction improvement CFS and acomplex B between said labeled analogue and reaction improvement CFS.

In the above description, “before these solutions are uniformly mixed”means “before each zone (liquid-liquid interface) of a solutionincluding a sample having an analyte and a labeled analogue (or asolution including a sample having an analyte, a labeled analogue andnot less than one kind of CFSs), and a solution including not less thanone kind of reaction improvement CFSs, along with if necessary theliquid, arranged in a capillary by a step (1) are uniformly mixed bymolecular diffusion”. In this connection, “interface” means the same asdescribed above.

In addition, in the above-described step (2), “concentrating saidanalyte and labeled analogue, and/or not less than one kind of reactionimprovement CFS, by applying a voltage onto a capillary” means that, assimilarly described above, said analyte and labeled analogue and/or notless than one kind of reaction improvement CFS gather in band-like(plug-like) on application of a voltage onto a capillary. In otherwords, it means that said substances gather on application of a voltageonto a capillary so that such a portion is generated whereinconcentration of said substances becomes higher than a substance in azone arranged in a step (1), namely, it means that an analyte and alabeled analogue, and/or not less than one kind of reaction improvementCFS garher on application of a voltage onto a capillary, and a portionis generated wherein concentration of an analyte and a labeled analogue,and/or concentration of a reaction improvement CFS becomes higher thanthat of an analyte and a labeled analogue, and/or not less than one kindof a reaction improvement CFS in a solution zone [for example, a zone ofa solution including a sample having an analyte and a labeled analogue(or a solution including a sample having an analyte, a labeled analogueand not less than one kind of CFSs), a zone of a solution includingreaction improvement CFSs] arranged in a step (1).

In the above step (2), “said analyte and labeled analogue areelectrophoretically contacted with said reaction improvement CFS(namely, said analyte is electrophoretically contacted with saidreaction improvement CFS, and said labeled analogue iselectrophoretically contacted with said reaction improvement CFS)” meansthat, similarly as described above, contact of said analyte, labeledanalogue and reaction improvement CFS are conducted (contact of each ofsaid analyte and reaction improvement CFS, and labeled analogue andreaction improvement CFS are contacted), not by (not depending on)molecular diffusion and by utilization of the phenomenon that when asolution containing a substance with higher electrophoretic mobility(faster electrophoretic speed) is arranged at upstream of a solutioncontaining a substance with lower electrophoretic mobility (slowelectrophoretic speed), and the electrophoresis is conducted, asubstance with higher electrophoretic mobility (faster electrophoreticspeed) in a solution overtakes a substance with lower electrophoreticmobility (slow electrophoretic speed).

(j) A Case when a Labeled Analogue, a CFS and a Reaction Improvement CFSare Used.

(1) (a) A solution containing a sample having an analyte and a labeledanalogue (or a solution including a sample having an analyte, a labeledanalogue and not less than one kind of CFSs), (b) a solution includingnot less than one kind of a CFS and (c) a solution including not lessthan one kind of a reaction improvement CFS are introduced and arrangedinto a capillary so that a zone of the solution containing a samplehaving an analyte and a labeled analogue (or the solution containing asample having an analyte, a labeled analogue and not less than one kindof CFSs), a zone of the solution including not less than one kind ofCFSs and a zone of the solution including not less than one kind ofreaction improvement CFSs are separately formed (so that liquid-liquidinterface is formed), and a complex A between said analyte, CFS andreaction improvement CFS and a complex B between said labeled analogue,CFS and reaction improvement CFS are formed on application of a voltageonto a capillary, without mixing these solutions in advance outside acapillary.

(2) Subsequently, said analyte and labeled analogue areelectrophoretically contacted with said CFS and reaction improvement CFS(namely, said analyte is electrophoretically contacted with said CFS andreaction improvement CFS, and said labeled analogue iselectrophoretically contacted with said CFS and reaction improvementCFS) while concentrating at least one selected from said analyte andlabeled analogue, not less than one kind of a CFS and not less than onekind of a reaction improvement CFS by applying a voltage onto saidcapillary before uniformly mixing these solutions, not by (not dependingon) molecular diffusion and without physically mixing, to form thecomplex A between said analyte, CFS and reaction improvement CFS and acomplex B between said labeled analogue, CFS and reaction improvementCFS.

In the above description, “before these solutions are uniformly mixed”means “before each zone (liquid-liquid interface) of a solutionincluding a sample having an analyte and a labeled analogue (or asolution including a sample having an analyte, a labeled analogue andnot less than one kind of CFSs), a solution including not less than onekind of CFSs, and a solution including not less than one kind ofreaction improvement CFSs, along with if necessary the liquid, arrangedin a capillary by a step (1) are uniformly mixed by moleculardiffusion”. In this connection, “interface” means the same as describedabove.

In addition, in the above-described step (2), “concentrating at leastone selected from said analyte and labeled analogue, not less than onekind of CFS and not less than one kind of reaction improvement CFS, byapplying a voltage onto a capillary” means that, as similarly describedabove, at least one selected from said analyte and labeled analogue, notless than one kind of CFS and not less than one kind of reactionimprovement CFS gather in band-like (plug-like) on application of avoltage onto a capillary. In other words, it means that said substancesgather on application of a voltage onto a capillary so that such aportion is generated wherein concentration of said substances becomeshigher than a substance in a zone arranged in a step (1), namely, itmeans that at least one selected from an analyte and labeled analogue,not less than one kind of CFS and not less than one kind of reactionimprovement CFS garher on application of a voltage onto a capillary, anda portion is generated wherein concentration of an analyte and a labeledanalogue, concentration of not less than one kind of a CFS orconcentration of not less than one kind of a reaction improvement CFSbecomes higher than that of an analyte and a labeled analogue, not lessthan one kind of a CFS or not less than one kind of a reactionimprovement CFS in a solution zone [for example, a zone of a solutionincluding an analyte and a labeled analogue (or a solution including asample having an analyte, a labeled analogue and not less than one kindof CFSs), a zone of a solution including not less than one kind of CFSs,and a zone of a solution including not less than one kind of reactionimprovement CFSs] arranged in a step (1).

In the above step (2), “said analyte and labeled analogue areelectrophoretically contacted with said CFS and reaction improvement CFS(namely, said analyte is electrophoretically contacted with said CFS andreaction improvement CFS, and said labeled analogue iselectrophoretically contacted with said CFS and reaction improvementCFS)” means that, similarly as described above, contact of said analyteand labeled analogue, CFS and reaction improvement CFS are conducted(namely, contact of each of said analyte, CFS and reaction improvementCFS, and said labeled analogue, CFS and reaction improvement CFS areconducted), not by (not depending on) molecular diffusion and byutilization of the phenomenon that when a solution containing asubstance with higher electrophoretic mobility (faster electrophoreticspeed) is arranged at upstream of a solution containing a substance withlower electrophoretic mobility (slow electrophoretic speed), a substancewith higher electrophoretic mobility (faster electrophoretic speed) in asolution overtakes a substance with lower electrophoretic mobility (slowelectrophoretic speed).

(k) A Case when a Reaction Improvement Analogue and a Labeled CFS areUsed.

(1) (a) A solution containing sample having an analyte and reactionimprovement analogue (or a solution including a sample having ananalyte, a reaction improvement analogue and not less than one kind ofCFSs) and (b) a solution including not less than one kind of labeledCFSs are introduced and arranged into a capillary so that a zone of asolution including the sample having an analyte and a reactionimprovement analogue (or the solution including a sample having ananalyte, a reaction improvement analogue and not less than one kind ofCFSs), and a zone of the solution including not less than one kind oflabeled CFSs are separately formed (so that liquid-liquid interface isformed), and a complex A between said analyte and labeled CFS and acomplex B between said reaction improvement analogue and labeled CFS areformed on application of a voltage onto a capillary, without mixingthese solutions in advance outside a capillary.

(2) Subsequently, said analyte and reaction improvement analogue areelectrophoretically contacted with said labeled CFS (namely, saidanalyte is electrophoretically contacted said labeled CFS, and saidreaction improvement analogue is electrophoretically contacted with saidlabeled CFS) while concentrating said analyte and reaction improvementanalogue and/or not less than one kind of labeled CFS by applying avoltage onto said capillary, before uniformly mixing these solutions,not by (not depending on) molecular diffusion and without physicallymixing, to form the complex A between said analyte and labeled CFS andthe complex B between said reaction improvement analogue and labeledCFS.

In the above description, “before these solutions are uniformly mixed”means “before each zone (liquid-liquid interface) of a solutionincluding a sample having an analyte and a reaction improvement analogue(or a solution including a sample having an analyte, a reactionimprovement analogue and not less than one kind of CFSs), and a solutionincluding not less than one kind of labeled CFSs, along with ifnecessary the liquid, arranged in a capillary by a step (1) areuniformly mixed by molecular diffusion”. In this connection, “interface”means the same as described above.

In addition, in the above-described step (2), “concentrating saidanalyte and reaction improvement analogue and/or not less than one kindof labeled CFS, by applying a voltage onto a capillary” means that, assimilarly described above, said analyte and reaction improvementanalogue and/or not less than one kind of labeled CFS gather inband-like (plug-like) on application of a voltage onto a capillary. Inother words, it means that said substances gather on application of avoltage onto a capillary so that such a portion is generated whereinconcentration of said substances becomes higher than a substance in azone arranged in a step (1), namely, it means that an analyte andreaction improvement analogue and/or not less than one kind of labeledCFS garher on application of a voltage onto a capillary, and a portionis generated wherein concentration of an analyte and a reactionimprovement analogue and/or concentration of not less than one kind of alabeled CFS becomes higher than that of an analyte and a reactionimprovement analogue, and/or not less than one kind of a labeled CFS ina solution zone [for example, a zone of a solution including an analyteand a reaction improvement analogue (or a solution including a samplehaving an analyte, a reaction improvement analogue and not less than onekind of CFSs), a solution including not less than one kind of labeledCFSs] arranged in a step (1).

In the above step (2), “said analyte and reaction improvement analogueare electrophoretically contacted with said labeled CFS (namely, saidanalyte is electrophoretically contacted said labeled CFS, and saidreaction improvement analogue is electrophoretically contacted with saidlabeled CFS)” means that, similarly as described above, contacted s saidanalyte, reaction improvement analogue and labeled CFS are conducted(namely, contact of each of said analyte and labeled CFS, and saidreaction improvement analogue and labeled CFS are conducted), not by(not depending on) molecular diffusion and by utilization of thephenomenon that when a solution containing a substance with higherelectrophoretic mobility (faster electrophoretic speed) is arranged atupstream of a solution containing a substance with lower electrophoreticmobility (slow electrophoretic speed), a substance with higherelectrophoretic mobility (faster electrophoretic speed) in a solutionovertakes a substance with lower electrophoretic mobility (slowelectrophoretic speed).

(l) A Case when a Labeled Analogue and a CFS are Used.

(1) (a) A solution including a sample having an analyte, and not lessthan one kind of CFSs, and (b) a solution containing a labeled analogueare introduced and arranged into a capillary so that a zone of asolution including a sample having an analyte, and not less than onekind of CFSs, and a zone of a solution including a labeled analogue areseparately formed (so that liquid-liquid interface is formed), and acomplex B between said labeled analogue and CFS is formed on applicationof a voltage onto a capillary, without mixing these solutions in advanceoutside a capillary.

(2) Subsequently, said labeled analogue is electrophoretically contactedwith CFS (namely, said labeled analogue is electrophoretically contactedwith said CFS not involved in the formation of a complex (complex A)with said analyte in the said solution including a sample having ananalyte, and not less than one kind of CFSs) while concentrating saidlabeled analogue and/or said CFS not involved in the formation of acomplex A by applying a voltage onto said capillary before uniformlymixing these solutions, not by (not depending on) molecular diffusionand without physically mixing, to form the complex B between saidlabeled analogue and CFS.

In the above description, “before these solutions are uniformly mixed”means “before each zone (liquid-liquid interface) of a solutionincluding a sample having an analyte, and not less than one kind ofCFSs, and a solution including a labeled analogue, along with ifnecessary the liquid, arranged in a capillary by a step (1) areuniformly mixed by molecular diffusion”. In this connection, “interface”means the same as described above.

In addition, in the above-described step (2), “concentrating saidlabeled analogue and/or said CFS not involved in the formation of acomplex A, by applying a voltage onto a capillary” means that, assimilarly described above, said labeled analogue and/or said CFS notinvolved in the formation of a complex A gather in band-like (plug-like)on application of a voltage onto a capillary. In other words, it meansthat said substances gather on application of a voltage onto a capillaryso that such a portion is generated wherein concentration of saidsubstances becomes higher than a substance in a zone arranged in a step(1), namely, it means that said labeled analogue and/or said CFS notinvolved in the formation of a complex A garher on application of avoltage onto a capillary, and a portion is generated whereinconcentration of a concentration of a labeled analogue and/or aconcentration of a CFS not involved in the formation of a complex Abecomes higher than that of a labeled analogue and/or a CFS not involvedin the formation of a complex A in a solution zone [for example, a zoneof a solution including a sample having an analyte, and not less thanone kind of CFSs, and a zone of a solution including a labeled analogue]arranged in a step (1).

In the above step (2), “said labeled analogue is electrophoreticallycontacted with CFS (namely, said labeled analogue is electrophoreticallycontacted with said CFS not involved in the formation of the complex(complex A) with said analyte in the said solution including a samplehaving an analyte, and not less than one kind of CFSs)” means that,similarly as described above, contact of said labeled analogue and CFSare conducted (namely, contact of said labeled analogue and said CFS notinvolved in the formation of the complex (complex A) with said analytein the said solution including a sample having an analyte, and not lessthan one kind of CFSs) not by (not depending on) molecular diffusion andby utilization of the phenomenon that when a solution containing asubstance with higher electrophoretic mobility (faster electrophoreticspeed) is arranged at upstream of a solution containing a substance withlower electrophoretic mobility (slow electrophoretic speed), a substancewith higher electrophoretic mobility (faster electrophoretic speed) in asolution overtakes a substance with lower electrophoretic mobility (slowelectrophoretic speed).

(m) A Case when a Labeled Analogue and a Reaction Improvement CFS areUsed.

(1) (a) A solution containing a sample having an analyte, and not lessthan one kind of reaction improvement CFSs, and (b) a solutioncontaining a labeled analogue are introduced and arranged into acapillary so that a zone of a solution including a sample having ananalyte, and not less than one kind of reaction improvement CFSs, and azone of a solution including a labeled analogue are separately formed(so that liquid-liquid interface is formed), and a complex B betweensaid labeled analogue and reaction improvement CFS is formed onapplication of a voltage onto a capillary, without mixing thesesolutions in advance outside a capillary.

(2) Subsequently, said labeled analogue is electrophoretically contactedwith said reaction improvement CFS (namely, said labeled analogue iselectrophoretically contacted with said reaction improvement CFS notinvolved in the formation of a complex (complex A) with said analyte inthe said solution including a sample having an analyte, and not lessthan one kind of reaction improvement CFS) while concentrating saidlabeled analogue and/or said reaction improvement CFS not involved inthe formation of a complex A by applying a voltage onto said capillarybefore uniformly mixing these solutions, not by (not depending on)molecular diffusion and without physically mixing, to form the complex Bbetween said labeled analogue and reaction improvement CFS.

In the above description, “before these solutions are uniformly mixed”means “before each zone (liquid-liquid interface) of a solutionincluding a sample having an analyte, and not less than one kind ofreaction improvement CFSs, and a solution including a labeled analogue,along with if necessary the liquid, arranged in a capillary by a step(1) are uniformly mixed by molecular diffusion”. In this connection,“interface” means the same as described above.

In addition, in the above-described step (2), “concentrating saidlabeled analogue and/or said reaction improvement CFS not involved inthe formation of a complex A, by applying a voltage onto a capillary”means that, as similarly described above, said labeled analogue and/orsaid reaction improvement CFS not involved in the formation of a complexA gather in band-like (plug-like) on application of a voltage onto acapillary. In other words, it means that said substances gather onapplication of a voltage onto a capillary so that such a portion isgenerated wherein concentration of said substances becomes higher than asubstance in a zone arranged in a step (1), namely, it means that alabeled analogue and/or a reaction improvement CFS not involved in theformation of a complex A garher on application of a voltage onto acapillary, and a portion is generated wherein concentration of a labeledanalogue and/or concentration of a reaction improvement CFS not involvedin the formation of a complex A becomes higher than that of a labeledanalogue and/or a reaction improvement CFS not involved in the formationof a complex A in a solution zone [for example, a zone of a solutionincluding a sample having an analyte, and not less than one kind ofreaction improvement CFSs, and a zone of a solution including a labeledanalogue] arranged in a step (1).

In the above step (2), “said labeled analogue is electrophoreticallycontacted with said reaction improvement CFS (namely, said labeledanalogue is electrophoretically contacted with said reaction improvementCFS not involved in the formation of a complex (complex A) with saidanalyte in the said solution including a sample having an analyte, andnot less than one kind of reaction improvement CFS)” means that,similarly as described above, contact of said labeled analogue andreaction improvement CFS are conducted (namely, contacted said labeledanalogue and said reaction improvement CFS not involved in the formationof the complex (complex A) with said analyte in the said solutionincluding a sample having an analyte, and not less than one kind ofreaction improvement CFS) not by (not depending on) molecular diffusionand by utilization of the phenomenon that when a solution containing asubstance with higher electrophoretic mobility (faster electrophoreticspeed) is arranged at upstream of a solution containing a substance withlower electrophoretic mobility (slow electrophoretic speed), a substancewith higher electrophoretic mobility (faster electrophoretic speed) in asolution overtakes a substance with lower electrophoretic mobility (slowelectrophoretic speed).

(n) A Case when a Labeled Analogue, a CFS and a Reaction Improvement CFSare Used.

(1) (a) A solution containing a sample having an analyte and not lessthan one kind of a CFS (or not less than one kind of a reactionimprovement CFS), (b) a solution containing a labeled analogue and (c) asolution containing not less than one kind of a reaction improvement CFS(or not less than one kind of a CFS) are introduced and arranged into acapillary so that a zone of the solution including a sample having ananalyte and at least one kind of a CFS (or not less than one kind of areaction improvement CFS), a zone of the solution including a labeledanalogue and a zone of a solution including not less than one kind of areaction improvement CFS (or not less than one kind of a CFS) areseparately formed (so that liquid-liquid interface is formed), and acomplex A between said analyte, CFS and reaction improvement CFS and acomplex B between said labeled analogue, CFS and reaction improvementCFS are formed on application of a voltage onto a capillary, withoutmixing these solutions in advance outside a capillary.

(2) Subsequently, said analyte and labeled analogue areelectrophoretically contacted with said CFS and reaction improvement CFS[namely, a comple between said analyte and CFS (or reaction improvementCFS) in said solution (a) is electrophoretically contacted with saidreaction improvement CFS (or CFS) in said solution (c), and said labeledanalogue is electrophoretically contacted with said CFS (or reactionimprovement CFS) not involved in the formation of said complex with saidanalyte in said solution (a) and said reaction improvement CFS (or CFS)in said solution (c)] while concentrating at least one selected from thecomplex A between said analyte and not less than one kind of CFS (or notless than one kind of reaction improvement CFS), labeled analogue, andnot less than one kind of reaction improvement CFS (or not less than onekind of CFS) by applying a voltage onto said capillary before uniformlymixing these solutions, not depending on molecular diffusion and withoutphysically mixing, to form the complex A between said analyte, CFS andreaction improvement CFS and the complex B between said labeledanalogue, CFS and reaction improvement CFS.

In the above description, “before these solutions are uniformly mixed”means “before each zone (liquid-liquid interface) of a solutionincluding a sample having an analyte and not less than one kind of a CFS(or not less than one kind of a reaction improvement CFS), a solutionincluding a labeled analogue and a solution including not less than onekind of reaction improvement CFSs (or not less than one kind of a CFS),along with if necessary the liquid, arranged in a capillary by a step(1) are uniformly mixed by molecular diffusion”. In this connection,“interface” means the same as described above.

In addition, in the above-described step (2), “concentrating at leastone selected from said complex between said analyte and not less thanone kind of a CFS (or not less than one kind of a reaction improvementCFS), said labeled analogue, and not less than one kind of a reactionimprovement CFS (or not less than one kind of a CFS), by applying avoltage onto a capillary” means that, as similarly described above, atleast one selected from said complex between said analyte and not lessthan one kind of a CFS (or not less than one kind of a reactionimprovement CFS), said labeled analogue, and not less than one kind of areaction improvement CFS (or not less than one kind of a CFS) gather inband-like (plug-like) on application of a voltage onto a capillary. Inother words, it means that said substances gather on application of avoltage onto a capillary so that such a portion is generated whereinconcentration of said substances becomes higher than a substance in azone arranged in a step (1), namely, it means that at least one selectedfrom a complex between said analyte and not less than one kind of a CFS(or not less than one kind of a reaction improvement CFS), a labeledanalogue, and not less than one kind of a reaction improvement CFS (ornot less than one kind of a CFS) garher on application of a voltage ontoa capillary, and a portion is generated wherein concentration of acomplex between said analyte and not less than one kind of a CFS (or notless than one kind of a reaction improvement CFS), concentration of alabeled analogue or concentration of not less than one kind of areaction improvement CFS (or not less than one kind of a CFS) becomeshigher than that of a complex between said analyte and at least one kindof a CFS (or not less than one kind of a reaction improvement CFS), alabeled analogue, or not less than one kind of a reaction improvementCFS (or not less than one kind of a CFS) in a solution zone [forexample, a zone of a solution including a sample having an analyte andnot less than one kind of a CFS (or not less than one kind of a reactionimprovement CFS), a zone of a solution including a labeled analogue anda zone of a solution including not less than one kind of reactionimprovement CFSs (or not less than one kind of a CFS)] arranged in astep (1).

In the above step (2), “said analyte and labeled analogue areelectrophoretically contacted with said CFS and reaction improvement CFS[namely, a comple between said analyte and CFS (or reaction improvementCFS) in said solution (a) is electrophoretically contacted with saidreaction improvement CFS (or CFS) in said solution (c), and said labeledanalogue is electrophoretically contacted with said CFS (or reactionimprovement CFS) not involved in the formation of said complex with saidanalyte in said solution (a) and said reaction improvement CFS (or CFS)in said solution (c)]” means that, similarly as described above,contacted said analyte, labeled analogue, CFS and reaction improvementCFS are conducted [namely, contact of said comple between said analyteand CFS (or reaction improvement CFS) in said solution (a) and saidreaction improvement CFS (or CFS) in said solution (c), and said labeledanalogue, said CFS (or reaction improvement CFS) not involved in theformation of said complex with said analyte in said solution (a) andsaid reaction improvement CFS (or CFS) in said solution (c) areconducted], not by (not depending on) molecular diffusion and byutilization of the phenomenon that when a solution containing asubstance with higher electrophoretic mobility (faster electrophoreticspeed) is arranged at upstream of a solution containing a substance withlower electrophoretic mobility (slow electrophoretic speed), a substancewith higher electrophoretic mobility (faster electrophoretic speed) in asolution overtakes a substance with lower electrophoretic mobility (slowelectrophoretic speed).

(o) A Case when a Reaction Improvement Analogue and a Labeled CFS areUsed.

(1) (a) A solution containing a sample having an analyte, and not lessthan one kind of labeled CFSs, and (b) a solution containing a reactionimprovement analogue are introduced and arranged into a capillary sothat a zone of a solution including a sample having an analyte, and notless than one kind of labeled CFSs, and a zone of a solution including areaction improvement analogue are separately formed (so thatliquid-liquid interface is formed), and a complex B between saidreaction improvement analogue and labeled CFS is formed on applicationof a voltage onto a capillary, without mixing these solutions in advanceoutside a capillary.

(2) Subsequently, said reaction improvement analogue iselectrophoretically contacted with labeled CFS (namely, said reactionimprovement analogue is electrophoretically with said labeled CFS notinvolved in the formation of a complex (complex A) with said analyte inthe said solution including a sample having an analyte, and not lessthan one kind of labeled CFSs) while concentrating said reactionimprovement and/or said labeled CFS not involved in the formation of acomplex A analogue by applying a voltage onto said capillary beforeuniformly mixing these solutions, not by (not depending on) moleculardiffusion and without physically mixing, to form the complex B betweensaid reaction improvement analogue and labeled CFS.

In the above description, “before these solutions are uniformly mixed”means “before each zone (liquid-liquid interface) of a solutionincluding a sample having an analyte, and not less than one kind oflabeled CFSs, and a solution including a reaction improvement analogue,along with if necessary the liquid, arranged in a capillary by a step(1) are uniformly mixed by molecular diffusion”. In this connection,“interface” means the same as described above.

In addition, in the above-described step (2), “concentrating a reactionimprovement analogue and/or a labeled CFS not involved in the formationof a complex A, by applying a voltage onto a capillary” means that, assimilarly described above, a reaction improvement analogue and/or alabeled CFS not involved in the formation of a complex A gather inband-like (plug-like) on application of a voltage onto a capillary. Inother words, it means that said substances gather on application of avoltage onto a capillary so that such a portion is generated whereinconcentration of said substances becomes higher than a substance in azone arranged in a step (1), namely, it means that said reactionimprovement analogue and/or said labeled CFS not involved in theformation of a complex A garher on application of a voltage onto acapillary, and a portion is generated wherein concentration ofconcentration of a reaction improvement analogue and/or concentration ofa labeled CFS not involved in the formation of a complex A becomeshigher than that of a reaction improvement analogue and/or a labeled CFSnot involved in the formation of a complex A in a solution zone [forexample, a zone of a solution including an analyte, and not less thanone kind of labeled CFSs, and a zone of a solution including a reactionimprovement analogue] arranged in a step (1).

In the above step (2), “said reaction improvement analogue iselectrophoretically contacted with labeled CFS (namely, said reactionimprovement analogue is electrophoretically with said labeled CFS notinvolved in the formation of a complex (complex A) with said analyte inthe said solution including a sample having an analyte, and not lessthan one kind of labeled CFSs)” means that, similarly as describedabove, contact of said reaction improvement analogue and labeled CFS areconducted (namely, contact of said reaction improvement analogue andsaid labeled CFS not involved in the formation of a complex (complex A)with said analyte in the said solution including a sample having ananalyte, and not less than one kind of labeled CFSs is conducted not by(not depending on) molecular diffusion and by utilization of thephenomenon that when a solution containing a substance with higherelectrophoretic mobility (faster electrophoretic speed) is arranged atupstream of a solution containing a substance with lower electrophoreticmobility (slow electrophoretic speed), a substance with higherelectrophoretic mobility (faster electrophoretic speed) in a solutionovertakes a substance with lower electrophoretic mobility (slowelectrophoretic speed).

In the above-described methods (a) to (o), as for level (degree) ofconcentration of a substance to be concentrated [for example, ananalyte, a CFS, a labeled CFS, a reaction improvement CFS, a labeledreaction improvement CFS, a labeled analogue, a reaction improvementanalogue, a complex between an analyte and a CFS, a complex between ananalyte and a reaction improvement substance, a complex between ananalyte and a labeled CFS, a complex between a labeled analogue and CFSand a complex between a reaction improvement analogue and CFS, etc.],concentration of a substance [for example, an analyte, a CFS, a labeledCFS, a reaction improvement CFS, a labeled reaction improvement CFS, alabeled analogue, a reaction improvement analogue, a complex between ananalyte and a CFS, a complex between an analyte and a reactionimprovement substance, a complex between an analyte and a labeled CFS, acomplex between a labeled analogue and CFS and a complex between areaction improvement analogue and CFS, etc.] at an gathered part (inband-like) of said substance on application of a voltage onto acapillary, relative to concentration of an substance [for example, ananalyte, a CFS, a labeled CFS, a reaction improvement CFS, a labeledreaction improvement CFS, a labeled analogue, a reaction improvementanalogue, a complex between an analyte and a CFS, a complex between ananalyte and a reaction improvement substance, a complex between ananalyte and a labeled CFS, a complex between a labeled analogue and CFSand a complex between a reaction improvement analogue and CFS, etc.] ina solution zone arranged by a step (1) is, as lower limit, usually notlower than 1.5 times, preferably not lower than 5 times, more preferablynot lower than 10 times, and further preferably not lower than 25 times,and upper limit is not especially limited, however usually not higherthan 10⁷ times, preferably not higher than 10⁶ times and more preferablynot higher than 10⁵ times.

In addition, in the above-described methods (a) to (o), “contacted whileconcentrating” means, similarly as described above, both cases whenconcentration and contact are simultaneously carried out, and a casewhen contact is carried out after concentration is substantiallycompleted, and therefore encompasses so-called all the cases other thana case when concentration is carried out after contact is substantiallycompleted.

In the above-described methods (a) to (o), a step (2) can be carriedout, similarly as described above, before a solution arranged in acapillary by a step (1) is uniformly mixed under conditions enablingconcentration, contacting and formation of a complex, by applying avoltage onto said capillary.

In the above-described methods (a) to (o), a step (2) can be carriedout, similarly as described above by applying a voltage onto saidcapillary under condition that concentration, contacting and formationof a complex can be carried out by applying a voltage onto saidcapillary, before a solution arranged in a capillary by a step (1) isuniformly mixed.

Specific examples, preferable embodiments, and the like of suchconditions are as described above, and for example, the above step (2)may be carried out in accordance with the above-described method forconcentration, in suitable consideration of electrophoretic mobility ofan analyte, a CFS, a labeled CFS, a reaction improvement CFS, a labeledreaction improvement CFS, a labeled analogue, a reaction improvementanalogue, a complex composed of 2 or more kinds thereof, to be used, orelectric conductivity of solutions including these.

In addition, applied voltage and other reaction conditions (for example,pH, temperature, time, etc.), and the like in a step (2) may alsosuitably be determined from a range as described above, in considerationof an analyte, a CFS, a labeled CFS, a reaction improvement CFS, alabeled reaction improvement CFS, a labeled analogue, a reactionimprovement analogue, a complex composed of 2 or more kinds thereof,solutions including these, and the like.

2. A Method for Separation of the Present Invention

A method for separation of the present invention features in electricalseparation of a complex between an analyte or an analogue thereof andnot less than one kind of CFSs, formed by a method for formation of acomplex of the present invention as described above, and a CFS notinvolved in formation of said complex or an analogue not involved information of said complex.

Namely, the features is that a complex between an analyte or an analoguethereof and a CFS in a solution, formed by each contact byelectrophoretically moving (migrating) in a capillary in a step (2) ofthe present invention, and a CFS not involved in formation of saidcomplex or an analogue not involved in formation of said complex arefurther electrophoretically moved (migrated) and are separated.

A method for separation of the present invention may be carried out inaccordance with a known method itself except in electrically separatinga complex between an analyte or an analogue thereof and not less thanone kind of CFSs, and a CFS not involved in formation of said complex oran analogue not involved in formation of said complex, in a known methoditself for separating a substance by electric movement (migration)using, for example, a capillary, and as for material and reagents to beused also, those used in known methods themselves may be used.

Therefore, a method for separation of the present invention includes thefollowing steps (1) to (3):

(1) a step (a step of introduction) of arranging (a) a solutioncontaining an analyte or an analogue thereof and (b) a solutioncontaining not less than one kind of a substance formable the complex(the CFS) with said analyte or said analogue thereof, in a capillary, sothat by applying a voltage to said capillary the complex between saidanalyte or said analogue thereof and the CFS are formed without mixingthese solutions in advance;

(2) a step (a step of concentrating reaction) of contacting said analyteor said analogue thereof with the CFS while concentrating said analyteor said analogue thereof and/or at least one kind of the CFSs byapplying a voltage to said capillary before uniformly mixing thesesolutions to form the complex between said analyte or said analoguethereof and the CFS; and

(3) a step (a step of separation) of separating said complex and the CFSnot involved in the formation of said complex or the analogue notinvolved in the formation of said complex by further electrical movement(migration).

In this connection, in the above description, embodiments, a specificexample, a preferable example, and the like of an analyte, an analogue(a labeled analogue, a reaction improvement analogue, etc.), a solutioncontaining an analyte or an analogue thereof, a sample including ananalyte, a CFS (a labeled CFS, a reaction improvement CFS, a labeledreaction improvement CFS, etc.), solutions including these, a step ofintroduction [a step (1)], a step of a concentrating reaction [a step(2)], are as described above.

2-1. A Step of Separation [a Step (3)]

As described above, a complex between an analyte or an analogue thereofand a CFS, obtained by steps (1) and (2) of the present invention, and aCFS not involved in formation of said complex or an analogue notinvolved in formation of said complex, are separated in a capillary, byfurther electrical movement (migration).

A method for separation of the present invention is for separation of acomplex between an analyte or an analogue thereof and a CFS, and a CFSnot involved in formation of said complex or an analogue not involved information of said complex, and in more specifically, (1) for separationof a complex between said analyte and CFS, and a CFS not involved information of said complex, by further electrical movement (migration);or (2) for separation of a complex between said analogue and CFS, and ananalogue not involved in formation of said complex or a complex betweensaid analyte and CFS not involved in formation of said complex, byfurther electrical movement (migration).

For example, when a method for separation of the present invention isused in a non-competitive method (for example, methods (a) to (g) to bedescribed later, and the like), and when a CFS including a labelingsubstance (a labeled CFS or a labeled reaction improvement CFS) is used,separation of at least a CFS including a labeling substance not involved(free labeling substances) in formation of a complex between an analyteand a CFS, and a complex including an analyte is enough. Thoughseparation of a CFS not including a labeling substance, from saidcomplex is not necessarily required, separation of said complex and allof the CFSs not involved (free CFSs) in formation of a complex ispreferable.

In addition, for example, when a method for separation of the presentinvention is used in a competitive method (for example, methods (h) to(o) to be described later, and the like), and when a labeled analogue isused, separation of at least a (final) complex between a labeledanalogue and (all of the) CFSs, and a labeled analogue not involved(free labeled analogue) in formation of said complex is enough.Separation of a complex between an analyte and a CFS, and a (final)complex between a labeled analogue and (all of the) CFSs is notnecessarily required. In addition, when a reaction improvement analogueis used, separation of at least a complex between a reaction improvementanalogue and a labeled CFS, and a complex between an analyte and alabeled CFS is enough.

A step (3) of the present invention may be carried out using a methodwhich enables to sufficiently separate a complex between an analyte oran analogue thereof and a CFS, and a CFS not involved in formation ofsaid complex or an analogue not involved in formation of said complex.As such a method, a known electrophoresis method itself and usually usedin this field can be used.

Specifically, electrophoresis methods based on various principles(separation modes) can be used. Examples of such method are ITP, IF, asdescribed above; so-called a capillary zone electrophoresis method (CZE)for separation of an objective substance by moving each substance indifferent speed depending on intensity of a charge thereof, wherein acapillary is fundamentally filled with only a buffer solution forelectrophoresis, [Reference: H. Hisamoto at al., Chem. Commun., (2001),2662, and the like]; so-called a micelle electro kinetic chromatography(MEKC) using a charged substance forming an ionic micelle, andseparating a objective substance by interaction with said micelle,[Reference: S. Terabe, Trends Anal. Chem., (1989), 8, 129, and thelike]; so-called a capillary gel electrophoresis method (CGE) forseparating an objective substance by using a filler such as a polymerhaving molecular sieve effect, and by charge of a molecule and size of amolecule inducing interaction with a polymer, [Reference: S. Hjerten, J.Chromatogr., (1987), 397, 409, and the like].

In this connection, in the present invention, reagents, and the likeused in an electrophoresis method as described above can be used, asappropriate. In addition, these reagents, an operation method inseparation, conditions, and the like can suitably be selected inaccordance with the description in references, as described above, andthe like.

As an electrophoresis method used in a step (3) of the presentinvention, any of an electrophoresis method based on the same principle(separation mode) as in a concentration method used in a step (2), or anelectrophoresis method based on different principle (separation mode) asin a concentration method used in a step (2) may be used.

In this connection, when use of an electrophoresis method based on thesame principle (separation mode) as in a step (2) provides insufficientseparation of a complex between an analyte or an analogue thereof and aCFS, and a CFS not involved in formation of said complex or an analoguenot involved in formation of said complex, use of an electrophoresismethod based on different principle (separation mode) as in aconcentration method used in a step (2) to carry out a step (3) of thepresent invention is desirable.

In such a case, execution of ITP, FASS, and the like in a step (2), andsubsequently CZE, and the like in a step (3) is particularly preferable.

As described above, in a separation method of the present invention, thefollowing 2 cases are included: (i) contact of said analyte or analoguethereof and CFS is conducted while concentrating an analyte or ananalogue thereof and/or at least one kind of a CFS, to form a complexbetween said analyte or analogue thereof and CFS by a step (2) (in otherwords, contact of said analyte or analogue thereof and CFS is conductedby applying a voltage onto said capillary under such condition as ananalyte or an analogue thereof and/or at least one kind of a CFS areconcentrated, to form a complex between said analyte or analogue thereofand CFS), and subsequently by using the same separation mode withoutchanging applied voltage (in other words, while applying a voltage withthe same intensity under the same condition as in a step (2)), saidcomplex and CFS not involved in formation of said complex or analoguenot involved in formation of said complex are separated by furtherelectric moving (migrating); (ii) or contact of said analyte or analoguethereof and CFS is conducted while concentrating an analyte or ananalogue thereof and/or at least one kind of a CFS, to form a complexbetween said analyte or analogue thereof and CFS by a step (2) (in otherwords, contact of said analyte or analogue thereof and CFS is conductedby applying a voltage onto said capillary under such condition as ananalyte or an analogue thereof and/or at least one kind of a CFS areconcentrated, to form a complex between said analyte or analogue thereofand CFS), and subsequently by using different separation mode and/ordifferent applied voltage [in other words, by changing condition(separation mode used) from that in a step (2) and/or intensity ofvoltage to be applied], said complex and a CFS not involved in formationof said complex or an analogue not involved in formation of said complexare separated by further electric moving (migrating).

As for applied voltage in a step (3), any range may be adopted as longas a complex between an analyte or an analogue thereof and a CFS, and aCFS not involved in formation of said complex or an analogue notinvolved in formation of said complex are sufficiently separated, and itis suitably selected from a range usually used in this field. In morespecifically, the voltage is applied so that electric field intensity isin a range of usually, as lower limit, not lower than 5 V/cm, preferablynot lower than 10 V/cm, more preferably not lower than 50 V/cm, furtherpreferably not lower than 500 V/cm, particularly preferably not lowerthan 1000 V/cm, and as upper limit, usually not higher than 10000 V/cm,preferably not higher than 5000 V/cm, and more preferably not higherthan 2000 V/cm. In this connection, as described above, applied voltagein a step (3) may be the same as or different from that in a step (2).

In addition, other separation conditions (for example, pH, temperature,time, and the like) may be any range as long as a complex of an analyteor an analogue thereof and a CFS, and a CFS not involved in formation ofsaid complex or an analogue not involved in formation of said complexare sufficiently separated, and it is suitably selected in accordancewith a known method itself usually used in this field.

Specifically, although not simply described due to dependency onproperty of an analyte or an analogue thereof and a CFS, however, lowerlimit of the pH is usually not lower than 2, preferably not lower than4, and more preferably not lower than 5, and upper limit is not higherthan 13, preferably not higher than 11 and more preferably not higherthan 9. Lower limit of the temperature is usually not lower than 0° C.,preferably not lower than 5° C. and more preferably not lower than 10°C., and upper limit is usually not higher than 90° C., preferably nothigher than 80° C., more preferably not higher than 50° C., furtherpreferably not higher than 40° C., and particularly preferably nothigher than 30° C. In addition, lower limit of the time is usually notshorter than 1 minute, preferably not shorter than 2 minutes and morepreferably not shorter than 3 minutes, and upper limit is not longerthan 20 minutes and more preferably not longer than 10 minutes.

As described above, a step (3) is carried out in a capillary, and assuch a capillary, the same one as used in a step (2) is included, andmaterial and inner diameter of the capillary are also as describedabove.

In addition, a step (3) of the present invention is carried out usuallyin a state that an electrophoresis medium such as a buffer solution forelectrophoresis or said buffer solution for electrophoresis containingfillers, is filled in a capillary (in separation region as describedabove. In this connection, a specific example, use concentration, pH,molecular weight, viscosity, an introduction method into a capillary,introduction timing, and the like of an electrophoresis medium are thesame as described above.

In this connection, as described above, when a step (3) of the presentinvention is carried out using an electrophoresis method based ondifferent principle (separation mode) from that in a concentrationmethod used in a step (2), an electrophoresis medium used in a step (2)and an electrophoresis medium used in a step (3) are not required to bethe same, and a different electrophoresis medium may be used by suitableselection thereof.

In this connection, steps (2) and (3) of the present invention areusually carried out using the same capillary (in the same capillary).Namely, a capillary used in a separation method of the present inventionhas at least a part enabling to carry out a step (1) of the presentinvention, a part enabling to carry out a step (2) of the presentinvention, and a part enabling to carry out a step (3) of the presentinvention. These parts may be present each independently in a capillary,or a part of or all of these parts may be present in overlapped state.In other words, as a result, a capillary used in a separation method ofthe present invention is one, which enables to arrange in a capillary(a) a solution containing an analyte or an analogue thereof and (b) asolution containing not less than one kind of substances formable acomplex (a CFS) with said analyte or analogue thereof, so that byapplying a voltage onto said capillary the complex between said analyteor analogue thereof and CFS is formed without mixing these solutions inadvance, and enables to contact said analyte or analogue thereof and CFSwhile concentrating said analyte or analogue thereof and/or at least onekind of CFS by applying a voltage onto said capillary, before uniformlymixing these solutions, to form the complex between said analyte oranalogue thereof and CFS, and further enables to separate said complexand a CFS not involved in formation of said complex or an analogue notinvolved in formation of said complex, by further electrical movement(migration?).

2-2. Specific Methods for Separation

Modes for carrying out the methods for separation of the presentinvention are specifically shown below.

(a) A Case when a CFS not Bound with a Labeling Substance and a ReactionImprovement Substance are Used.

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs] and (b) a solutioncontaining not less than one kind of CFSs are introduced and arrangedinto a capillary, as the above-described step (1) in the case (a) of“1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte is electrophoretically contacted withsaid CFS to form the complex between said analyte and the CFS, as theabove-described step (2) in the case (a) of “1-6. Specific methods forforming a complex”, and

(3) Said complex and CFS not involved in formation of said complex areseparated in a separation region of a capillary by further electricmovement (migration).

(b) A Case when a Labeled CFS is Used.

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs] and (b) a solutioncontaining not less than one kind of labeled CFSs are introduced andarranged into a capillary, as the above-described step (1) in the case(b) of “1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte is electrophoretically contacted withsaid labeled CFS to form the complex between said analyte and labeledCFS, as the above-described step (2) in the case (b) of “1-6. Specificmethods for forming a complex”, and

(3) Said complex and said labeled CFS not involved in formation of saidcomplex are separated in a separation region of a capillary by furtherelectric movement (migration).

(c) A Case when a Reaction Improvement CFS is Used.

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs] and (b) a solutioncontaining not less than one kind of reaction improvement CFSs areintroduced and arranged into a capillary, as the above-described step(1) in the case (c) of “1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte is electrophoretically contacted withsaid reaction improvement CFS to form the complex between said analyteand reaction improvement CFS, as the above-described step (2) in thecase (c) of “1-6. Specific methods for forming a complex”, and

(3) Said complex and said reaction improvement CFS not involved information of said complex are separated in a separation region of acapillary by further electric movement (migration).

(d) A Case when a Labeled Reaction Improvement CFS is Used.

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs] and (b) a solutioncontaining not less than one kind of labeled reaction improvement CFSsare introduced and arranged into a capillary, as the above-describedstep (1) in the case (d) of “1-6. Specific methods for forming acomplex”,

(2) Subsequently, said analyte is electrophoretically contacted saidlabeled reaction improvement CFS to form the complex between saidanalyte and labeled reaction improvement CFS, as the above-describedstep (2) in the case (d) of “1-6. Specific methods for forming acomplex”, and

(3) Said complex and said labeled reaction improvement CFS not involvedin formation of said complex are separated in a separation region of acapillary by further electric movement (migration).

(e) A Case when a CFS not Bound with a Labeling Substance and a ReactionImprovement Substance, and a Reaction Improvement CFS are Used.

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs], (b) a solution containingnot less than one kind of a CFS and (c) a solution containing not lessthan one kind of a reaction improvement CFS are introduced and arrangedinto a capillary, as the above-described step (1) in the case (e) of“1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte is electrophoretically contacted withsaid CFS and reaction improvement CFS to form the complex between saidanalyte, CFS and reaction improvement CFS, as the above-described step(2) in the case (e) of “1-6. Specific methods for forming a complex”,and

(3) Said complex and said CFS not involved in formation of said complexand optionally a reaction improvement CFS not involved in formation ofsaid complex are separated in a separation region of a capillary byfurther electric movement (migration).

(f) A Case when a Labeled CFS and a Reaction Improvement CFS are Used.

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs], (b) a solution containingnot less than one kind of a labeled CFS and (c) a solution containingnot less than one kind of a reaction improvement CFS are introduced andarranged into a capillary, as the above-described step (1) in the case(f) of “1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte is electrophoretically contacted withsaid labeled CFS and reaction improvement CFS to form the complexbetween said analyte, labeled CFS and reaction improvement CFS, as theabove-described step (2) in the case (f) of “1-6. Specific methods forforming a complex”, and

(3) Said complex and said labeled CFS not involved in formation of saidcomplex and optionally a reaction improvement CFS not involved information of said complex are separated in a separation region of acapillary by further electric movement (migration).

(g) A Case when a CFS not Bound with a Labeling Substance and a ReactionImprovement Substance, and a Labeled Reaction Improvement CFS are Used.

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs], (b) a solution containingnot less than one kind of a CFS and (c) a solution containing not lessthan one kind of a labeled reaction improvement CFS are introduced andarranged into a capillary, as the above-described step (1) in the case(g) of “1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte is electrophoretically contacted withsaid CFS and labeled reaction improvement CFS to form the complexbetween said analyte, CFS and labeled reaction improvement CFS, as theabove-described step (2) in the case (g) of “1-6. Specific methods forforming a complex”, and

(3) Said complex and said labeled reaction improvement CFS not involvedin formation of said complex and optionally a CFS not involved information of said complex are separated in a separation region of acapillary by further electric movement (migration).

A method for separation of the present invention can also be used inso-called a competitive method. The procedure in carrying it out in acompetitive method is as follows:

(h) A Case when a Labeled Analogue and a CFS are Used.

(1) (a) A solution containing a sample having an analyte and a labeledanalogue (or a solution including a sample having an analyte, a labeledanalogue and not less than one kind of CFSs) and (b) a solutioncontaining not less than one kind of CFSs are introduced and arrangedinto a capillary, as the above-described step (1) in the case, (h) of“1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte and labeled analogue areelectrophoretically contacted with said CFS (namely, said analyte iselectrophoretically contacted with said CFS, and said labeled analogueelectrophoretically contacted with said CFS) to form the complex Abetween said analyte and CFS and a complex B between said labeledanalogue and CFS, as the above-described step (2) in the case (h) of“1-6. Specific methods for forming a complex”, and

(3) Said complex B and said labeled analogue not involved in formationof said complex B are separated in a separation region of a capillary byfurther electric movement (migration).

(i) A Case when a Labeled Analogue and a Reaction Improvement CFS areUsed.

(1) (a) A solution including a sample having an analyte and a labeledanalogue (or a solution including a sample having an analyte, a labeledanalogue and not less than one kind of CFSs) and (b) a solutioncontaining not less than one kind of reaction improvement CFSs areintroduced and arranged into a capillary, as the above-described step(1) in the case (i) of “1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte and labeled analogue areelectrophoretically contacted with said reaction improvement CFS(namely, said analyte is electrophoretically contacted with saidreaction improvement CFS, and said labeled analogue iselectrophoretically contacted with said reaction improvement CFS) toform the complex A between said analyte and reaction improvement CFS anda complex B between said labeled analogue and reaction improvement CFS,as the above-described step (2) in the case (i) of “1-6. Specificmethods for forming a complex”, and

(3) Said complex B and said labeled analogue not involved in formationof said complex B are separated in a separation region of a capillary byfurther electric movement (migration).

(j) A Case when a Labeled Analogue, a CFS and a Reaction Improvement CFSare Used.

(1) (a) A solution containing a sample having an analyte and a labeledanalogue (or a solution including a sample having an analyte, a labeledanalogue and not less than one kind of CFSs), (b) a solution includingnot less than one kind of a CFS and (c) a solution including not lessthan one kind of a reaction improvement CFS are introduced and arrangedinto a capillary, as the above-described step (1) in the case (j) of“1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte and labeled analogue areelectrophoretically contacted with said CFS and reaction improvement CFS(namely, said analyte is electrophoretically contacted with said CFS andreaction improvement CFS, and said labeled analogue iselectrophoretically contacted with said CFS and reaction improvementCFS) to form the complex A between said analyte, CFS and reactionimprovement CFS and a complex B between said labeled analogue, CFS andreaction improvement CFS, as the above-described step (2) in the case(j) of “1-6. Specific methods for forming a complex”, and

(3) Said complex B and said labeled analogue not involved in formationof said complex B are separated in a separation region of a capillary byfurther electric movement (migration).

(k) A Case when a Reaction Improvement Analogue and a Labeled CFS areUsed.

(1) (a) A solution containing sample having an analyte and reactionimprovement analogue (or a solution including a sample having ananalyte, a reaction improvement analogue and not less than one kind ofCFSs) and (b) a solution including not less than one kind of labeledCFSs are introduced and arranged into a capillary, as theabove-described step (1) in the case (k) of “1-6. Specific methods forforming a complex”,

(2) Subsequently, said analyte and reaction improvement analogue areelectrophoretically contacted with said labeled CFS (namely, saidanalyte is electrophoretically contacted said labeled CFS, and saidreaction improvement analogue is electrophoretically contacted with saidlabeled CFS) to form the complex A between said analyte and labeled CFSand the complex B between said reaction improvement analogue and labeledCFS, as the above-described step (2) in the case (k) of “1-6. Specificmethods for forming a complex”, and

(3) Said complex B and said complex A are separated in a separationregion of a capillary by further electric movement (migration).

(l) A Case when a Labeled Analogue and a CFS are Used.

(1) (a) A solution including a sample having an analyte, and not lessthan one kind of CFSs, and (b) a solution containing a labeled analogueare introduced and arranged into a capillary, as the above-describedstep (1) in the case (1) of “1-6. Specific methods for forming acomplex”,

(2) Subsequently, said labeled analogue is electrophoretically contactedwith CFS (namely, said labeled analogue is electrophoretically contactedwith said CFS not involved in the formation of a complex (complex A)with said analyte in the said solution including a sample having ananalyte, and not less than one kind of CFSs) to form the complex Bbetween said labeled analogue and CFS, as the above-described step (2)in the case (1) of “1-6. Specific methods for forming a complex”, and

(3) Said complex B and said labeled analogue not involved in formationof said complex B are separated in a separation region of a capillary byfurther electric movement (migration).

(m) A Case when a Labeled Analogue and a Reaction Improvement CFS areUsed.

(1) (a) A solution containing a sample having an analyte, and not lessthan one kind of reaction improvement CFSs, and (b) a solutioncontaining a labeled analogue are introduced and arranged into acapillary, as the above-described step (1) in the case (m) of “1-6.Specific methods for forming a complex”,

(2) Subsequently, said labeled analogue is electrophoretically contactedwith said reaction improvement CFS (namely, said labeled analogue iselectrophoretically contacted with said reaction improvement CFS notinvolved in the formation of a complex (complex A) with said analyte inthe said solution including a sample having an analyte, and not lessthan one kind of reaction improvement CFS) to form the complex B betweensaid labeled analogue and reaction improvement CFS, as theabove-described step (2) in the case (m) of “1-6. Specific methods forforming a complex”, and

(3) Said complex B and said labeled analogue not involved in formationof said complex are separated in a separation region of a capillary byfurther electric movement (migration).

(n) A Case when a Labeled Analogue, a CFS and a Reaction Improvement CFSare Used.

(1) (a) A solution containing a sample having an analyte and not lessthan one kind of a CFS (or not less than one kind of a reactionimprovement CFS), (b) a solution containing a labeled analogue and (c) asolution containing not less than one kind of a reaction improvement CFS(or not less than one kind of a CFS) are introduced and arranged into acapillary, as the above-described step (1) in the case (n) of “1-6.Specific methods for forming a complex”,

(2) Subsequently, said analyte and labeled analogue areelectrophoretically contacted with said CFS and reaction improvement CFS[namely, a comple between said analyte and CFS (or reaction improvementCFS) in said solution (a) is electrophoretically contacted with saidreaction improvement CFS (or CFS) in said solution (c), and said labeledanalogue is electrophoretically contacted with said CFS (or reactionimprovement CFS) not involved in the formation of said complex with saidanalyte in said solution (a) and said reaction improvement CFS (or CFS)in said solution (c)], as the above-described step (2) in the case (n)of “1-6. Specific methods for forming a complex”, and

(3) Said complex B and said labeled analogue not involved in formationof said complex B are separated in a separation region of a capillary byfurther electric movement (migration).

(o) A Case when a Reaction Improvement Analogue and a Labeled CFS areUsed.

(1) (a) A solution containing a sample having an analyte, and not lessthan one kind of labeled CFSs, and (b) a solution containing a reactionimprovement analogue are introduced and arranged into a capillary, asthe above-described step (1) in the case (o) of “1-6. Specific methodsfor forming a complex”,

(2) Subsequently, said reaction improvement analogue iselectrophoretically contacted with labeled CFS (namely, said reactionimprovement analogue is electrophoretically with said labeled CFS notinvolved in the formation of a complex (complex A) with said analyte inthe said solution including a sample having an analyte, and not lessthan one kind of labeled CFSs) to form the complex B between saidreaction improvement analogue and labeled CFS, as the above-describedstep (2) in the case (o) of “1-6. Specific methods for forming acomplex”, and

(3) Said complex B and said complex A are separated in a separationregion of a capillary by further electric movement (migration).

3. A Method for Measuring of the Present Invention

By measuring the amount of a complex between an analyte or an analoguethereof and a CFS, and the amount of a CFS not involved in formation ofsaid complex or an analogue not involved in formation of said complex,which are separated by a method for separation of the present invention,by a method corresponding to property of, for example, a labelingsubstance in a complex, or a labeling substance in a CFS or analoguethereof not involved in formation of a complex, the amount of an analytepresent in a sample can be determined simply, in high sensitivity and ina short time.

Therefore, a method for measuring of the present invention features incomprising:

(1) a step (a step of introduction) of arranging (a) a solutioncontaining an analyte or an analogue thereof and (b) a solutioncontaining not less than one kind of a substance formable the complex(the CFS) with said analyte or said analogue thereof, in a capillary, sothat by applying a voltage to said capillary the complex between saidanalyte or said analogue thereof and the CFS are formed without mixingthese solutions in advance;

(2) a step (a step of concentrating reaction) of contacting said analyteor said analogue thereof with the CFS while concentrating said analyteor said analogue thereof and/or at least one kind of the CFSs byapplying a voltage to said capillary before uniformly mixing thesesolutions to form the complex between said analyte or said analoguethereof and the CFS;

(3) a step (a step of separation) of separating said complex and the CFSnot involved in the formation of said complex or the analogue notinvolved in the formation of said complex by further electrical movement(migration); and

(4) a step (a step of measurement) of measuring the amount of thusseparated complex, or the amount of the CFS not involved in formation ofsaid complex or the amount of the analogue not involved in formation ofsaid complex, to determine the amount of an analyte based on the result.

In this connection, in the above description, embodiments, a specificexample, a preferable example, and the like of an analyte, an analogue(a labeled analogue, a reaction improvement analogue, etc.), a solutioncontaining an analyte or an analogue thereof, a sample including ananalyte, a CFS (a labeled CFS, a reaction improvement CFS, a labeledreaction improvement CFS, etc.), solutions including these, a step ofintroduction [a step (1)], a step of a concentrating reaction [a step(2)], a step of separation [a step (3)] are as described above.

A method for measuring of the present invention is for measuring theamount of a complex between an analyte or an analogue thereof and a CFS,or the amount of a CFS not involved in formation of said complex or theamount of an analogue not involved in formation of said complex, whichare separated by a step (3) of the present invention as described above,and for determining the amount of an analyte, based on the results, andin more specifically, (1) for measuring the amount of a complex betweenan analyte and a CFS, or the amount of a CFS not involved in formationof said complex, which are separated by a step (3) of the presentinvention, and for determining the amount of an analyte, based on theresults; or (2) for measuring the amount of a complex between ananalogue and a CFS, or the amount of an analogue not involved information of said complex, or the amount of a complex between an analyteand a CFS, which are separated, and for determining the amount of ananalyte, based on the results.

A method for measuring of the present invention is applicable to any ofa non-competitive method or a competitive method.

Namely, when a method for measuring of the present invention is carriedout by a non-competitive method, for example, it may be carried out asfollows:

(1) By a step (1) of the present invention, (a) A solution containing ananalyte [for example, (i) a sample including an analyte, (ii) a solutionincluding a sample having an analyte, and not less than one kind ofCFSs] and (b) a solution containing not less than one kind of CFSs (aCFS, a labeled CFS, a reaction improvement CFS, a labeled reactionimprovement CFS, combinations thereof) are arranged into a capillary, sothat by applying a voltage to said capillary the complex between saidanalyte and the CFS [(analyte-CFS) complex, (analyte-labeled CFS)complex, (analyte-reaction improvement CFS) complex, (analyte-labeledreaction improvement CFS) complex, (CFS-analyte-reaction improvementCFS) complex, (labeled CFS-analyte-reaction improvement CFS) complex,(CFS-analyte-labeled reaction improvement CFS) complex, and combinationsthereof, etc.] is formed, without mixing these solutions in advance;

(2) by a step (2) of the present invention, said analyte is contactedwith said CFS while concentrating said analyte and/or at least one kindof the CFSs by applying a voltage to said capillary before uniformlymixing these solutions to form the complex between said analyte and theCFS;

(3) by a step (3) of the present invention, said complex and a CFS notinvolved in formation of said complex are separated by furtherelectrical movement (migration); and

(4) the amount of separated complex or the amount of a CFS not involvedin formation of said complex is measured to determine the amount of ananalyte in a sample based on the result.

In addition, when a method for measuring of the present invention iscarried out by a competitive method, for example, it may be carried outas follows:

(1) By a step (1) of the present invention, (i) (a) a sample includingan analyte (or a solution including a sample having an analyte, and notless than one kind of CFSs), (b) a solution containing a labeledanalogue labeled by a labeling substance (or a solution including alabeled analogue and not less than one kind of CFSs), and (c) a solutioncontaining not less than one kind of CFSs (a CFS, a reaction improvementCFS, combinations thereof), or (ii) (a) a solution containing a samplehaving an analyte, and a labeled analogue (or a solution including asample having an analyte, a labeled analogue and not less than one kindof CFSs), and (b) a solution containing not less than one kind of CFSs(a CFS, a reaction improvement CFS, combinations thereof), or (iii) (a)a solution including a sample having an analyte, and not less than onekind of CFSs (a CFS, a reaction improvement CFS, combinations thereof),and (b) a solution containing a labeled analogue (or a solutionincluding a labeled analogue and not less than one kind of CFSs), arearranged into a capillary, so that by applying a voltage to saidcapillary a complex B between said labeled analogue and CFS [(labeledanalogue-CFS) complex, (labeled analogue-reaction improvement CFS)complex, (CFS-labeled analogue-reaction improvement CFS) complex, andcombinations thereof, etc.] is formed, or a complex A between saidanalyte and CFS [(analyte-CFS) complex, (analyte-reaction improvementCFS) complex, (CFS-analyte-reaction improvement CFS) complex, andcombinations thereof, etc.] and such Complex B are formed, withoutmixing these solutions in advance;

(2) by a step (2) of the present invention, (i) said labeled analogue iscontacted with said CFS [namely, said labeled analogue is contacted witha CFS not involved in the formation of a complex (complex A) includingsaid analyte] or (ii) said analyte and labeled analogue are contactedwith said CFS (namely, said analyte is contacted with said CFS and saidlabeled analogue is contacted with said CFS) while concentrating atleast one of said analyte, said labeled analogue and not less than onekind of CFSs before uniformly mixing these solutions, to form thecomplex B between said labeled analogue and CFS or to form the complex Abetween said analyte and CFS and the complex B;

(3) by a, step (3) of the present invention, said complex B, and alabeled analogue not involved in formation of said complex B areseparated by further electrical movement (migration); and

(4) the amount of separated complex B, or the amount of said labeledanalogue not involved in formation of said complex B is measured todetermine the amount of an analyte in a sample based on the result.

In addition, when a method for measuring of the present invention iscarried out by a competitive method, for example, it may also be carriedout as follows:

(1) By a step (1) of the present invention, (i) (a) a sample includingan analyte (or a solution including a sample having an analyte, and notless than one kind of CFSs), (b) a solution containing a analogue boundwith a reaction improvement substance (a reaction improvement analogue)(or a solution including a reaction improvement analogue and not lessthan one kind of CFSs), and (c) a solution containing not less than onekind of CFSs (a CFS, a labeled CFS, combinations thereof), or (ii) (a) asolution including a sample having an analyte, and a reactionimprovement analogue (or a solution including a sample having ananalyte, a reaction improvement analogue and not less than one kind ofCFSs), and (b) a solution containing not less than one kind of CFSs (aCFS, a labeled CFS, combinations thereof), or (iii) (a) a solutionincluding a sample having an analyte, and not less than one kind of CFSs(a CFS, a labeled CFS, combinations thereof), and (b) a solutioncontaining a reaction improvement analogue (or a solution including areaction improvement analogue and not less than one kind of CFSs), arearranged into a capillary, so that by applying a voltage to saidcapillary a complex B between said reaction improvement analogue andlabeled CFS is formed, or a complex A between said analyte and labeledCFS and such Complex B are formed, without mixing these solutions inadvance;

(2) by a step (2) of the present invention, (i) said reactionimprovement analogue is contacted with said labeled CFS [namely, saidreaction improvement analogue is contacted with a labeled CFS notinvolved in the formation of a complex (complex A) including saidanalyte] or (ii) said analyte and reaction improvement analogue arecontacted with said labeled CFS (namely, said analyte is contacted withsaid labeled CFS and said reaction improvement analogue is contactedwith said labeled CFS) while concentrating at least one of said analyte,said reaction improvement analogue and not less than one kind of labeledCFS before uniformly mixing these solutions, to form the complex Bbetween said reaction improvement analogue and labeled CFS or to formthe complex A between said analyte and labeled CFS and the complex B;

(3) by a step (3) of the present invention, said complex B and complex Aare separated by further electrical movement (migration); and

(4) the amount of separated complex B or the amount of separated complexA is measured to determine the amount of an analyte in a sample based onthe result.

In this connection, an analogue (a labeled analogue or a reactionimprovement analogue) is used by (i) co-presence with an analyte in asample (namely, a sample including an analyte) as a solution including alabeled analogue or a reaction improvement analogue and an analyte (asolution containing an analyte and an analogue); or (2) withoutco-presence with an analyte in a sample (namely, a sample including ananalyte) and separately from a solution containing an analyte, as asolution containing an analogue.

3-1. A Step of Measurement [a Step (4)]

In a step (4) of the present invention, the amount of a complex or theamount of a CFS not involved in formation of said complex or the amountof an analogue not involved in formation of said complex, which areseparated, may be measured, for example, by a method corresponding toproperty of a labeling substance in said complex, or a labelingsubstance in a CFS not involved in formation of said complex or alabeling substance in an analogue not involved in formation of saidcomplex, and based on measurement results of said labeling substance.Namely, in a non-competitive method, the amount of a complex between ananalyte and a CFS, or the amount of a CFS not involved in formation ofsaid complex, which are separated, may be determined, for example, by amethod corresponding to property of a labeling substance in saidcomplex, or labeling substance in CFS not involved in formation of saidcomplex, and based on the result of measurement of said labelingsubstance. In a competitive method, the amount of a complex B or theamount of a labeled analogue not involved in formation of said complex B(or the amount of a complex B or the amount of a complex A), which areseparated, may be determined, by a method corresponding to property of alabeling substance in said complex B, or a labeling substance in alabeled analogue not involved in formation of said complex B (or alabeling substance in a complex A), and based on the measurement resultof said labeling substance.

Measurement of a labeling substance may be carried out in accordancewith each specified method corresponding to a kind of a labelingsubstance. For example, when said property is enzyme activity,measurement of a labeling substance may be carried out in accordancewith a common method such as EIA or a hybridization method [for example,a method described in “Enzyme immunoassay method, protein, nucleic acid,enzyme, separate vol., No. 31, Edited by T. Kitagawa, T. Nannbara, A.Tuji, 51 to 63, KYQRITSU SHUPPAN Co., Ltd., published on Sep. 10, 1987”,etc.]; when said property is radioactivity, measurement of a labelingsubstance may be carried out by using suitably selected measuringinstrument such as a liquid immersion type GM counter, a liquidscintillation counter, and a well-type scintillation counter, dependingon kind and intensity of radiation ray emitted by said radio activesubstance in accordance with a common method such as RIA or ahybridization method, [for example, Medical Chemistry ExperimentalCourse, vol. 8, Edited by U. Yamamura, 1^(st) Ed., published by NakayamaBookstore in 1971; Biochemistry Experimental Course 2, A TracerExperiment Method (part 2), A. Takemura, H, Honjo, 501 to 525, publishedby TOKYO KAGAKU DOJIN Co., Ltd. on Feb. 25, 1977]; when said property isfluorescence, measurement of a labeling substance may be carried out inaccordance with a common method such as FIA or a hybridization methodusing measuring instrument such as fluorospectrometer or a confocallaser scanning microscope [a method described in, for example,“Illustration Explanation, Fluorescent antibody, A. Kawao, 1^(st) Ed,published by Softscience Co., Ltd., 1983”; “Medical ChemistryExperimental Course, vol. 2, Chemistry of nucleic acid III, M.Saneyoshi, 299 to 318, published by TOKYO KAGAKU DOJIN Co., Ltd. on Dec.15, 1977”]; when said property is luminescence, measurement of alabeling substance may be carried out in accordance with a common methodusing measuring instrument such as a photon-counter [for example, amethod described in “Enzyme immunoassay method, protein, nucleic acid,enzyme, separate vol., No. 31, Edited by T. Kitagawa, T. Nannbara, A.Tuji, 252 to 263, KYORITSU SHUPPAN Co., Ltd., published on, Sep. 10,1987”]; further when said property is UV absorption, measurement of alabeling substance may be carried out in accordance with a common methodusing measuring instrument such as a spectrometer; when said property iscolor phenomenon, measurement of a labeling substance may be carried outin accordance with a common method using measuring instrument such as aspectrometer or a microscope; when said property is spin, measurement ofa labeling substance may be carried out in accordance with a commonmethod using electron spin resonance instrument [for example, a methoddescribed in “Enzyme immunoassay method, protein, nucleic acid, enzyme,separate vol., No. 31, Edited by T. Kitagawa, T. Nannbara, A. Tuji, 264to 271, KYORITSU SHUPPAN Co., Ltd., published on Sep. 10, 1987”, etc.].

In addition, determination of the amount of an analyte present in asample, based on measured amount of a complex or amount of a CFS notinvolved in formation of said complex or amount of an analogue notinvolved in formation of said complex, namely the amount of a labelingsubstance in a complex or amount of a labeling substance in a CFS notinvolved in formation of said complex or a labeling substance in ananalogue not involved in formation of said complex, may be carried outfor example, as follows:

In a competitive method, determination of the amount of an analytepresent in a sample based on the measured amount of a complex or theamount of a CFS not involved in formation of said complex, namely theamount of a labeling substance in a complex or the amount of a labelingsubstance in a CFS not involved in formation of said complex, obtainedas above, can be carried out, for example, by preparing a calibrationcurve showing relation between the amount of an analyte and the amountof a labeling substance in a complex or the amount of a labelingsubstance in a CFS not involved in formation of said complex, obtainedby measurement with a similar method using a sample containing a knownconcentration of an analyte, and by applying the amount of a labelingsubstance obtained by measurement of a sample containing an analyte tosaid calibration curve. In addition, in a non-competitive method,determination of the amount of an analyte present in a sample based onthe amount of a complex B or the amount of a labeled analogue notinvolved in formation of said complex B (or the amount of a complex B orthe amount of complex A), namely the amount of a labeling substance in acomplex B or the amount of a labeling substance in a labeled analoguenot involved in formation of said complex B (or the amount of a labelingsubstance in a complex A), obtained as above, can be carried out, forexample, by preparing a calibration curve showing relation between theamount of an analyte, and the amount of a labeling substance in acomplex B or the amount of a labeling substance in a labeled analoguenot involved in formation of said complex B (or the amount of a labelingsubstance in a complex A), obtained by measurement with a similar methodusing a sample containing a known concentration of an analyte, and byapplying the amount of a labeling substance obtained by measurement of asample containing an analyte to said calibration curve.

In addition, by the addition of a known concentration of a detectablesubstance as an internal standard into a sample, and by comparing theamount of said substance added as internal standard, with the amount ofa complex not involved in formation of said complex, or the amount of aCFS or the amount of an analogue not involved in formation of saidcomplex [namely, the amount of a labeling substance in a complex or theamount of a labeling substance in a CFS not involved in formation ofsaid complex, or the amount of a labeling substance in a complex B orthe amount of a labeling substance in a labeled analogue not involved information of said complex B (or the amount of a labeling substance in acomplex B or the amount of a labeling substance in a complex A)], therelative amount of an analyte in a sample may be calculated. Inaddition, such calculation can also be correct an error amongelectrophoresis equipment (device). Furthermore, by using the mobilityof a peak of an internal standard it is possible to also correct themobility of an objective peak.

Such detectable substances (internal standards) include, for example,peptide, protein, nucleic acid (DNA, RNA), an amino acid, sugar, a sugarchain, etc., labeled with the above described labeling substance; and afluorescent substance, etc.

In addition, in the present invention, when an enzyme is used as alabeling substance, and the like, substrates or other coupling enzymesof said enzyme may be required to measure activity of said enzyme. Insuch a case, for example, these substrates or other coupling enzymes maybe arranged in a capillary at the downstream side of a solutionincluding a complex or a CFS not involved in formation of said complexor an analogue not involved in formation of said complex [namely, acomplex between an analyte and a CFS or a CFS not involved in formationof said complex, or a complex B or a labeled analogue not involved information of said complex B (or a complex B or a complex A)], separatedby a step (3) of the present invention, at least before carrying out astep (4) of the present invention. It is preferable that in a step (1)of the present invention a solution including these substrates or othercoupling enzymes is arranged at further downstream side of the solution(zone) arranged at the most downstream side among a solution (zone)including an analyte or analogue thereof and a solution (zone) includingnot less than one kind of CFSs, and steps (1) to (4) of the presentinvention are carried out.

In addition, when an intercalator dye is used as a labeling substance,in a step (1) of the present invention said intercalator dye is notrequired to be introduced and arranged in a capillary with a solutioncontaining an analyte or an analogue thereof, and a solution includingnot less than one kind of CFSs. And also, in a step (2) of the presentinvention said intercalator dye is not required to make contact with ananalyte or an analogue thereof and a CFS. It is only necessary to carryout at least a step (3) of the present invention in the presence of saidintercalator dye. In such a case, specifically, for example, saidintercalator dye may be contained in an electrophoresis medium and/orbuffer solution used in a step (3) of the present invention. Amongothers, in the present invention, it is preferable that a step (2) and astep (3) of the present invention are carried out in the presence ofsaid intercalator dye. In this case, said intercalator dye may becontained in an electrophoresis medium and/or buffer solution used in astep (2) and in a step (3) of the present invention.

3-2. Use of a Charged Polymer

In the present invention, it is preferable that a step (2) is carriedout in the presence of a charged polymer.

Namely, (i) by making contact of an analyte or an analogue thereof, anda CFS in the presence of a charged polymer to form a complex betweensaid analyte or analogue thereof and CFS, or (ii) by making contact ofan analyte, an analogue (a labeled analogue) and a CFS in the presenceof a charged polymer to form a complex A between said analyte and CFS,and a complex B between said labeled analogue and CFS; or (iii) bymaking contact of an analyte, a reaction improvement analogue and alabeled CFS in the presence of a charged polymer to form a complex Abetween said analyte and labeled CFS and a complex B between saidreaction improvement analogue and labeled CFS, effect of a co-presentsubstance in a sample (particularly in a serum sample) having bad effecton analysis can be reduced. Specifically, for example, (i) by makingcontact of an analyte and CFS in the presence of a charged polymer, acomplex between said analyte and CFS [(analyte-CFS) complex,(analyte-labeled CFS) complex, (analyte-reaction improvement CFS)complex, (analyte-labeled reaction improvement CFS) complex,(CFS-analyte-reaction improvement CFS) complex, (labeledCFS-analyte-reaction improvement CFS) complex, (CFS-analyte-labeledreaction improvement CFS) complex, and combinations thereof, etc.] isformed; or (ii) by making contact of an analyte, a labeled analogue anda CFS in the presence of a charged polymer (namely, by making contact ofsaid analyte and CFS, and said labeling analogue and CFS), a complex Abetween said analyte and said CFS [(analyte-CFS) complex,(analyte-reaction improvement CFS) complex, (CFS-analyte-reactionimprovement CFS) complex, and combinations thereof, etc.] and a complexB between said labeled analogue and CFS [(labeled analogue-CFS) complex,(labeled analogue-reaction improvement CFS) complex, (CFS-labeledanalogue-reaction improvement CFS) complex, and combinations thereof,etc.] are formed; or (iii) by making contact of an analyte, a reactionimprovement analogue and a labeled CFS (namely, by making contact ofsaid analyte and labeled CFS, and said reaction improvement analogue andlabeled CFS) in the presence of a charged polymer, a complex A betweensaid analyte and said labeled CFS and a complex B between said reactionimprovement analogue and said labeled CFS are formed.

As a charged polymer used in the present invention, one having theopposite charge (plus or minus) from that of a co-present substance in asample is used. In addition, a charged polymer having the same charge asthat of a CFS used is preferable.

As such a charged polymer, polyanionic polymers and polycationicpolymers are included.

Polyanionic polymers include, polysaccharides such as heparin, heparinsulfate, chondroitin sulfate, dextran sulfate, polytungstic acid,tungstophosphoric acid, hyaluronic acid, dermatan sulfate andpolyanethole sulfate, etc.; polynucleotides such as DNA (plasmid DNA,calf thymus DNA, salmon sperm DNA, DNA bound with cellulose andsynthetic DNA, etc.), and RNA, etc.; polypeptides such as polyaminoacids (polyaspartic acid, polyglutamic acid, etc.), a syntheticpolypeptide, etc.; synthetic polymer compounds such as poly-dldC,polyvinyl sulfate, polyacrylic acid, etc.; ceramics such as glassparticle, colloidal glass, glass milk, etc.; and complexes thereof; andthe like.

In addition, polycationic polymers include, polysaccharides such aschitosan, derivatives thereof, etc.; polypeptides such as polylysine,polyhistidine, polyarginine, protamine, histone, ornithine, etc.;synthetic polymer compounds such as polyallyl amine, polyethylene imine,polyvinyl amine, etc.; polyamines such as spermine, spermidine, etc.;cationic lipid; ceramics; complexes thereof; and the like.

Among them, an anionic polysaccharide is preferable, and heparin sulfateis particularly preferable.

The above-described charged polymers may be used alone or in suitablecombination with two or more kinds.

A method for making the above described charged polymers present incarrying out a step (2) is not especially limited as long as formationof a complex can finally be carried out in the presence of a chargedpolymer.

Such a method includes, for example, a method for making a chargedpolymer co-present in an electrophoresis medium to be filled in acapillary as described above; a method for making a charged polymerco-present in a solution containing an analyte or an analogue thereof,and/or in a solution containing CFS; and the like.

Among these, the charged polymer is co-present preferably in a solution(zone) other than the solution (zone) containing an analyte, morepreferably in at least one solution (zone) arranged adjacently upstreamside or downstream side of the solution (zone) containing an analyte.

Use amount of the above-described charged polymer is not simply bedescribed due to dependency on the kind of the charged polymer used,however, for example, concentration in an electrophoresis medium to befilled in a channel is, as lower limit, usually not lower than 0.01%(w/v), preferably not lower than 0.05% (w/v), and more preferably notlower than 0.5% (w/v), and as upper limit, usually not higher than 50%(w/v), preferably not higher than 10% (w/v) and further preferably nothigher than 5% (w/v), and among others about 1% (w/v) is particularlypreferable. In addition, concentration in a solution containing ananalyte or an analogue thereof or in a solution containing a CFS is, aslower limit, usually not lower than 0.001% (w/v), preferably not lowerthan 0.01% (w/v), more preferably not lower than 0.02% (w/v), andfurther preferably not lower than 0.025% (w/v), and as upper limit,usually not higher than 10% (w/v), preferably not higher than 5% (w/v),more preferably not higher than 1% (w/v) and further preferably nothigher than 0.05% (w/v).

3-3. A Specific Methods for Measurement

Modes for carrying out the methods for measurement of the presentinvention are specifically shown below.

(1) A Non-Competitive Method

A method for measurement for the case of a non-competitive method is asfollows:

(a) A Case when a CFS not Bound with a Labeling Substance and a ReactionImprovement Substance are Used.

In this case, for example, it may be carried out as the following[method A] or [method B].

[Method A]:

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs] and (b) a solutioncontaining not less than one kind of CFSs are introduced and arrangedinto a capillary, as the above-described step (1) in the case (a) of“1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte is electrophoretically contacted withsaid CFS to form the complex between said analyte and the CFS, as theabove-described step (2) in the case (a) of “1-6. Specific methods forforming a complex”,

(3) Said complex and said CFS not involved in formation of said complexare separated by further electric movement (migration), as theabove-described step (3) in the case (a) of “2-2. specific methods forseparation”, and

(4) The amount of a CFS contained in the separated complex or the amountof a CFS not involved in formation of said complex is measured by amethod corresponding to property (kind) of a CFS, and the amount of ananalyte in a sample is calculated by applying the measurement result(measurement value) to a calibration curve showing relation between theamount of an analyte and the amount of a CFS, obtained by a similarmethod using a sample containing a known concentration of an analyte.

[Method B]:

A known concentration of a detectable substance as an internal standardis added to at least one of a solution containing an analyte and asolution containing CFS in the above-described step (1) of [method A],and the above-described steps (1) to (3) are carried out using thatsolution containing the internal standard. The amount of a CFS containedin the separated complex or the amount of a CFS not involved information of said complex is measured by a method corresponding toproperty (kind) of a CFS, and the amount of an analyte in a sample iscalculated by comparing the measurement result (measurement value) withthe amount of said substance added as internal standard.

(b) A Case when a Labeled CFS is Used.

In this case, for example, it may be carried out as the following[method A] or [method B].

[Method A]:

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs] and (b) a solutioncontaining not less than one kind of labeled CFSs are introduced andarranged into a capillary, as the above-described step (1) in the case(b) of “1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte is electrophoretically contacted withsaid labeled CFS to form the complex between said analyte and labeledCFS, as the above-described step (2) in the case (b) of “1-6. Specificmethods for forming a complex”,

(3) Said complex and said labeled CFS not involved in formation of saidcomplex are separated by further electric movement (migration) byfurther electric movement (migration), as the above-described step (3)in the case (b) of “2-2. specific methods for separation”, and

(4) The amount of a labeling substance contained in the separatedcomplex or the amount of a labeling substance contained in a labeled CFSnot involved in formation of said complex is measured by a methodcorresponding to property (kind) of a labeling substance, and the amountof an analyte in a sample is calculated by applying the measurementresult (measurement value) to a calibration curve showing relationbetween the amount of an analyte and the amount of a labeling substance,obtained by a similar method using a sample containing a knownconcentration of an analyte.

[Method B]:

A known concentration of a detectable substance as an internal standardis added to at least one of a solution containing an analyte and asolution containing a labeled CFS in the above-described step (1) of[method A], and the above-described steps (1) to (3) are carried outusing that solution containing the internal standard. The amount of alabeling substance contained in the separated complex or the amount of alabeling substance contained in a labeled CFS not involved in formationof said complex is measured by a method corresponding to property (kind)of a labeling substance, and the amount of an analyte in a sample iscalculated by comparing the measurement result (measurement value) withthe amount of said substance added as internal standard.

(c) A Case when a Reaction Improvement CFS is Used.

In this case, for example, it may be carried out as the following[method A] or [method B].

[Method A]:

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs] and (b) a solutioncontaining not less than one kind of reaction improvement CFSs areintroduced and arranged into a capillary, as the above-described step(1) in the case (c) of “1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte is electrophoretically contacted withsaid reaction improvement CFS to form the complex between said analyteand reaction improvement CFS, as the above-described step (2) in thecase (c) of “1-6. Specific methods for forming a complex”,

(3) Said complex and said reaction improvement CFS not involved information of said complex are by further electric movement (migration),as the above-described step (3) in the case (c) of “2-2. specificmethods for separation”, and

(4) The amount of a reaction improvement CFS contained in the separatedcomplex or the amount of a reaction improvement CFS not involved information of said complex is measured by a method corresponding toproperty (kind) of a reaction improvement CFS, and the amount of ananalyte in a sample is calculated by applying the measurement result(measurement value) to a calibration curve showing relation between theamount of an analyte and the amount of a reaction improvement CFS,obtained by a similar method using a sample containing a knownconcentration of an analyte.

[Method B]:

A known concentration of a detectable substance as an internal standardis added to at least one of a solution containing an analyte and asolution containing a reaction improvement CFS in the above-describedstep (1) of [method A], and the above-described steps (1) to (3) arecarried out using that solution containing the internal standard. Theamount of a reaction improvement CFS contained in the separated complexor the amount of a reaction improvement CFS not involved in formation ofsaid complex is measured by a method corresponding to property (kind) ofa reaction improvement CFS, and the amount of an analyte in a sample iscalculated by comparing the measurement result (measurement value) withthe amount of said substance added as internal standard.

(d) A Case when a Labeled Reaction Improvement CFS is Used.

In this case, for example, it may be carried out as the following[method A] or [method B].

[Method A]:

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs] and (b) a solutioncontaining not less than one kind of labeled reaction improvement CFSsare introduced and arranged into a capillary, as the above-describedstep (1) in the case (d) of “1-6. Specific methods for forming acomplex”,

(2) Subsequently, said analyte is electrophoretically contacted saidlabeled reaction improvement CFS to form the complex between saidanalyte and labeled reaction improvement CFS, as the above-describedstep (2) in the case (d) of “1-6. Specific methods for forming acomplex”,

(3) Said complex and said labeled reaction improvement CFS not involvedin formation of said complex are separated by further electric movement(migration), as the above-described step (3) in the case (d) of “2-2.specific methods for separation”, and

(4) The amount of a labeling substance contained in the separatedcomplex or the amount of a labeling substance contained in a labeledreaction improvement CFS not involved in formation of said complex ismeasured by a method corresponding to property (kind) of a labelingsubstance, and the amount of an analyte in a sample is calculated byapplying the measurement result (measurement value) to a calibrationcurve showing relation between the amount of an analyte and the amountof a labeling substance, obtained by a similar method using a samplecontaining a known concentration of an analyte.

[Method B]:

A known concentration of a detectable substance as an internal standardis added to at least one of a solution containing an analyte and asolution containing a labeled reaction improvement CFS in theabove-described step (1) of [method A], and the above-described steps(1) to (3) are carried out using that solution containing the internalstandard. The amount of a labeling substance contained in the separatedcomplex or the amount of a labeling contained substance in a labeledreaction improvement CFS not involved in formation of said complex ismeasured by a method corresponding to property (kind) of a labelingsubstance, and the amount of an analyte in a sample is calculated bycomparing the measurement result (measurement value) with the amount ofsaid substance added as internal standard.

(e) A Case when a CFS not Bound with a Labeling Substance and a ReactionImprovement Substance, and a Reaction Improvement CFS are Used.

In this case, for example, it may be carried out as the following[method A] or [method B].

[Method A]:

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs], (b) a solution containingnot less than one kind of a CFS and (c) a solution containing not lessthan one kind of a reaction improvement CFS are introduced and arrangedinto a capillary, as the above-described step (1) in the case (e) of“1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte is electrophoretically contacted withsaid CFS and reaction improvement CFS to form the complex between saidanalyte, CFS and reaction improvement CFS, as the above-described step(2) in the case (e) of “1-6. Specific methods for forming a complex”,

(3) Said complex and said CFS not involved in formation of said complexand optionally a reaction improvement CFS not involved in formation ofsaid complex are separated by further electric movement (migration), asthe above-described step (3) in the case (e) of “2-2. specific methodsfor separation”, and

(4) The amount of a CFS contained in the separated complex or the amountof a reaction improvement CFS contained in the separated complex, or theamount of a CFS not involved in formation of said complex or the amountof a reaction improvement CFS not involved in formation of said complexis measured by a method corresponding to property (kind) of a CFS or areaction improvement CFS, and the amount of an analyte in a sample iscalculated by applying the measurement result (measurement value) to acalibration curve showing relation between the amount of an analyte, andthe amount of a CFS or the amount of a reaction improvement CFS,obtained by a similar method using a sample containing a knownconcentration of an analyte.

[Method B]:

A known concentration of a detectable substance as an internal standardis added to at least one selected from a solution containing an analyte,a solution containing a CFS and a solution containing a reactionimprovement CFS in the above-described step (1) of [method A], and theabove-described steps (1) to (3) are carried out using that solutioncontaining the internal standard. The amount of a CFS contained in theseparated complex or the amount of a reaction improvement CFS containedin the separated complex, or the amount of a CFS not involved information of said complex or the amount of a reaction improvement CFSnot involved in formation of said complex is measured by a methodcorresponding to property (kind) of a CFS or a reaction improvement CFS,and the amount of an analyte in a sample is calculated by comparing themeasurement result (measurement value) with the amount of said substanceadded as internal standard.

(f) A Case when a Labeled CFS and a Reaction Improvement CFS are Used.

In this case, for example, it may be carried out as the following[method A] or [method B].

[Method A]:

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs], (b) a solution containingnot less than one kind of a labeled CFS and (c) a solution containingnot less than one kind of a reaction improvement CFS are introduced andarranged into a capillary, as the above-described step (1) in the case(f) of “1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte is electrophoretically contacted withsaid labeled CFS and reaction improvement CFS to form the complexbetween said analyte, labeled CFS and reaction improvement CFS, as theabove-described step (2) in the case (f) of “1-6. Specific methods forforming a complex”, and

(3) Said complex and said labeled CFS not involved in formation of saidcomplex and optionally a reaction improvement CFS not involved information of said complex are separated by further electric movement(migration), as the above-described step (3) in the case (f) of “2-2.specific methods for separation”, and

(4) The amount of a labeling substance contained in the separatedcomplex or the amount of a labeling substance contained in a labeled CFSnot involved in formation of said complex is measured by a methodcorresponding to property (kind) of a labeling substance, and the amountof an analyte in a sample is calculated by applying the measurementresult (measurement value) to a calibration curve showing relationbetween the amount of an analyte and the amount of a labeling substance,obtained by a similar method using a sample containing a knownconcentration of an analyte.

[Method B]:

A known concentration of a detectable substance as an internal standardis added to at least one selected from a solution containing an analyte,a solution containing a labeled CFS and a solution containing a reactionimprovement CFS in the above-described step (1) of [method A], and theabove-described steps (1) to (3) are carried out using that solutioncontaining the internal standard. The amount of a labeling substancecontained in the separated complex or the amount of a labeling substancecontained in a labeled CFS not involved in formation of said complex ismeasured by a method corresponding to property (kind) of a labelingsubstance, and the amount of an analyte in a sample is calculated bycomparing the measurement result (measurement value) with the amount ofsaid substance added as internal standard.

(g) A Case when a CFS not Bound with a Labeling Substance and a ReactionImprovement Substance, and a Labeled Reaction Improvement CFS are Used.

In this case, for example, it may be carried out as the following[method A] or [method B].

[Method A]:

(1) (a) A solution containing an analyte [for example, (i) a sampleincluding an analyte, (ii) a solution including a sample having ananalyte, and not less than one kind of CFSs], (b) a solution containingnot less than one kind of a CFS and (c) a solution containing not lessthan one kind of a labeled reaction improvement CFS are introduced andarranged into a capillary, as the above-described step (1) in the case(g) of “1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte is electrophoretically contacted withsaid CFS and labeled reaction improvement CFS to form the complexbetween said analyte, CFS and labeled reaction improvement CFS, as theabove-described step (2) in the case (g) of “1-6. Specific methods forforming a complex”,

(3) Said complex and said labeled reaction improvement CFS not involvedin formation of said complex and optionally a CFS not involved information of said complex are separated by further electric movement(migration), as the above-described step (3) in the case (g) of “2-2.specific methods for separation”, and

(4) The amount of a labeling substance contained in the separatedcomplex or the amount of a labeling substance contained in a labeledreaction improvement CFS not involved in formation of said complex ismeasured by a method corresponding to property (kind) of a labelingsubstance, and the amount of an analyte in a sample is calculated byapplying the measurement result (measurement value) to a calibrationcurve showing relation between the amount of an analyte and the amountof a labeling substance, obtained by a similar method using a samplecontaining a known concentration of an analyte.

[Method B]:

A known concentration of a detectable substance as an internal standardis added to at least one selected from a solution containing an analyte,a solution containing a CFS and a solution containing a labeled reactionimprovement CFS in the above-described step (1) of [method A], and theabove-described steps (1) to (3) are carried out using that solutioncontaining the internal standard. The amount of a labeling substancecontained in the separated complex or the amount of a labeling substancecontained in a labeled reaction improvement CFS not involved information of said complex is measured by a method corresponding toproperty (kind) of a labeling substance, and the amount of an analyte ina sample is calculated by comparing the measurement result (measurementvalue) with the amount of said substance added as internal standard.

(2) A Competitive Method

A method for measurement for the case of a competitive method is asfollows:

(h) A Case when a Labeled Analogue and a CFS are Used.

In this case, for example, it may be carried out as the following[method A] or [method B].

[Method A]:

(1) (a) A solution containing a sample having an analyte and a labeledanalogue (or a solution including a sample having an analyte, a labeledanalogue and not less than one kind of CFSs) and (b) a solutioncontaining not less than one kind of CFSs are introduced and arrangedinto a capillary, as the above-described step (1) in the case (h) of“1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte and labeled analogue areelectrophoretically contacted with said CFS (namely, said analyte iselectrophoretically contacted with said CFS, and said labeled analogueelectrophoretically contacted with said CFS) to form the complex Abetween said analyte and CFS and a complex B between said labeledanalogue and CFS, as the above-described step (2) in the case (h) of“1-6. Specific methods for forming a complex”,

(3) Said complex B and said labeled analogue not involved in formationof said complex B are separated by further electric movement(migration), as the above-described step (3) in the case (h) of “2-2.specific methods for separation”, and

(4) The amount of a labeling substance contained in the separatedcomplex B or the amount of a labeling substance contained in a labeledanalogue not involved in formation of said complex B is measured by amethod corresponding to property (kind) of a labeling substance, and theamount of an analyte in a sample is calculated by applying themeasurement result (measurement value) to a calibration curve showingrelation between the amount of an analyte and the amount of a labelingsubstance, obtained by a similar method using a sample containing aknown concentration of an analyte.

[Method B]:

A known concentration of a detectable substance as an internal standardis added to a solution containing a sample having an analyte and alabeled analogue (or a solution including a sample having an analyte, alabeled analogue and not less than one kind of CFSs) or a solutioncontaining a CFS in the above-described step (1) of [method A], and theabove-described steps (1) to (3) are carried out using that solutioncontaining the internal standard. The amount of a labeling substancecontained in the separated complex B or the amount of a labelingsubstance contained in a labeled analogue not involved in formation ofsaid complex B is measured by a method corresponding to property (kind)of a labeling substance, and the amount of an analyte in a sample iscalculated by comparing the measurement result (measurement value) withthe amount of said substance added as internal standard.

(i) A Case when a Labeled Analogue and a Reaction Improvement CFS areUsed.

In this case, for example, it may be carried out as the following[method A] or [method B].

[Method A]:

(1) (a) A solution including a sample having an analyte and a labeledanalogue (or a solution including a sample having an analyte, a labeledanalogue and not less than one kind of CFSs) and (b) a solutioncontaining not less than one kind of reaction improvement CFSs areintroduced and arranged into a capillary, as the above-described step(1) in the case (i) of “1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte and labeled analogue areelectrophoretically contacted with said reaction improvement CFS(namely, said analyte is electrophoretically contacted with saidreaction improvement CFS, and said labeled analogue iselectrophoretically contacted with said reaction improvement CFS) toform the complex A between said analyte and reaction improvement CFS anda complex B between said labeled analogue and reaction improvement CFS,as the above-described step (2) in the case (i) of “1-6. Specificmethods for forming a complex”

(3) Said complex B and said labeled analogue not involved in formationof said complex B are separated by further electric movement(migration), as the above-described step (3) in the case (i) of “2-2.specific methods for separation”, and

(4) The amount of a labeling substance contained in the separatedcomplex B or the amount of a labeling substance contained in a labeledanalogue not involved in formation of said complex B is measured by amethod corresponding to property (kind) of a labeling substance, and theamount of an analyte in a sample is calculated by applying themeasurement result (measurement value) to a calibration curve showingrelation between the amount of an analyte and the amount of a labelingsubstance, obtained by a similar method using a sample containing aknown concentration of an analyte.

[Method B]:

A known concentration of a detectable substance as an internal standardis added to a solution containing a sample having an analyte and alabeled analogue (or a solution including a sample having an analyte, alabeled analogue and not less than one kind of CFSs) or a solutioncontaining a reaction improvement CFS in the above-described step (1) of[method A], and the above-described steps (1) to (3) are carried outusing that solution containing the internal standard. The amount of alabeling substance contained in the separated complex B or the amount ofa labeling substance contained in a labeled analogue not involved information of said complex B is measured by a method corresponding toproperty (kind) of a labeling substance, and the amount of an analyte ina sample is calculated by comparing the measurement result (measurementvalue) with the amount of said substance added as internal standard.

(j) A Case when a Labeled Analogue, a CFS and a Reaction Improvement CFSare Used.

In this case, for example, it may be carried out as the following[method A] or [method B].

[Method A]:

(1) (a) A solution containing a sample having an analyte and a labeledanalogue (or a solution including a sample having an analyte, a labeledanalogue and not less than one kind of CFSs), (b) a solution includingnot less than one kind of a CFS and (c) a solution including not lessthan one kind of a reaction improvement CFS are introduced and arrangedinto a capillary, as the above-described step (1) in the case (j) of“1-6. Specific methods for forming a complex”,

(2) Subsequently, said analyte and labeled analogue areelectrophoretically contacted with said CFS and reaction improvement CFS(namely, said analyte is electrophoretically contacted with said CFS andreaction improvement CFS, and said labeled analogue iselectrophoretically contacted with said CFS and reaction improvementCFS) to form the complex A between said analyte, CFS and reactionimprovement CFS and a complex B between said labeled analogue, CFS andreaction improvement CFS, as the above-described step (2) in the case(j) of “1-6. Specific methods for forming a complex”,

(3) Said complex B and said labeled analogue not involved in formationof said complex B are separated by further electric movement(migration), as the above-described step (3) in the case (j) of “2-2.specific methods for separation”, and

(4) The amount of a labeling substance contained in the separatedcomplex B or the amount of a labeling substance contained in a labeledanalogue not involved in formation of said complex B is measured by amethod corresponding to property (kind) of a labeling substance, and theamount of an analyte in a sample is calculated by applying themeasurement result (measurement value) to a calibration curve showingrelation between the amount of an analyte and the amount of a labelingsubstance, obtained by a similar method using a sample containing aknown concentration of an analyte.

[Method B]:

A known concentration of a detectable substance as an internal standardis added to at least one selected from a solution containing a samplehaving an analyte and a labeled analogue (or a solution including asample having an analyte, a labeled analogue and not less than one kindof CFSs), a solution containing a CFS and a solution containing areaction improvement CFS in the above-described step (1) of [method A],and the above-described steps (1) to (3) are carried out using thatsolution containing the internal standard. The amount of a labelingsubstance contained in the separated complex B or the amount of alabeling substance contained in a labeled analogue not involved information of said complex B is measured by a method corresponding toproperty (kind) of a labeling substance, and the amount of an analyte ina sample is calculated by comparing the measurement result (measurementvalue) with the amount of said substance added as internal standard.

(k) A Case when a Reaction Improvement Analogue and a Labeled CFS areUsed.

In this case, for example, it may be carried out as the following[method A] or [method B].

[Method A]:

(1) (a) A solution containing sample having an analyte and reactionimprovement analogue (or a solution including a sample having ananalyte, a reaction improvement analogue and not less than one kind ofCFSs) and (b) a solution including not less than one kind of labeledCFSs are introduced and arranged into a capillary, as theabove-described step (1) in the case (k) of “1-6. Specific methods forforming a complex”,

(2) Subsequently, said analyte and reaction improvement analogue areelectrophoretically contacted with said labeled CFS (namely, saidanalyte is electrophoretically contacted said labeled CFS, and saidreaction improvement analogue is electrophoretically contacted with saidlabeled CFS) to form the complex A between said analyte and labeled CFSand the complex B between said reaction improvement analogue and labeledCFS, as the above-described step (2) in the case (k) of “1-6. Specificmethods for forming a complex”,

(3) Said complex B and said complex A are separated by further electricmovement (migration), as the above-described step (3) in the case (k) of“2-2. specific methods for separation”, and

(4) The amount of a labeling substance contained in the separatedcomplex B or the amount of a labeling substance contained in theseparated complex A is measured by a method corresponding to property(kind) of a labeling substance, and the amount of an analyte in a sampleis calculated by applying the measurement result (measurement value) toa calibration curve showing relation between the amount of an analyteand the amount of a labeling substance, obtained by a similar methodusing a sample containing a known concentration of an analyte.

[Method B]:

A known concentration of a detectable substance as an internal standardis added to a solution containing a sample having an analyte and areaction improvement analogue (or a solution including a sample havingan analyte, a reaction improvement analogue and not less than one kindof CFSs) or a solution containing a labeled CFS in the above-describedstep (1) of [method A], and the above-described steps (1) to (3) arecarried out using that solution containing the internal standard. Theamount of a labeling substance contained in the separated complex B orthe amount of a labeling substance contained in said the separatedcomplex A is measured by a method corresponding to property (kind) of alabeling substance, and the amount of an analyte in a sample iscalculated by comparing the measurement result (measurement value) withthe amount of said substance added as internal standard.

(l) A Case when a Labeled Analogue and a CFS are Used.

In this case, for example, it may be carried out as the following[method A] or [method B].

[Method A]:

(1) (a) A solution including a sample having an analyte, and not lessthan one kind of CFSs, and (b) a solution containing a labeled analogueare introduced and arranged into a capillary, as the above-describedstep (1) in the case (1) of “1-6. Specific methods for forming acomplex”,

(2) Subsequently, said labeled analogue is electrophoretically contactedwith CFS (namely, said labeled analogue is electrophoretically contactedwith said CFS not involved in the formation of a complex (complex A)with said analyte in the said solution including a sample having ananalyte, and not less than one kind of CFSs) to form the complex Bbetween said labeled analogue and CFS, as the above-described step (2)in the case (1) of “1-6. Specific methods for forming a complex”,

(3) Said complex B and said labeled analogue not involved in formationof said complex B are separated by further electric movement(migration), as the above-described step (3) in the case (1) of “2-2.specific methods for separation”, and

(4) The amount of a labeling substance contained in the separatedcomplex B or the amount of a labeling substance contained in a labeledanalogue not involved in formation of said complex B is measured by amethod corresponding to property (kind) of a labeling substance, and theamount of an analyte in a sample is calculated by applying themeasurement result (measurement value) to a calibration curve showingrelation between the amount of an analyte and the amount of a labelingsubstance, obtained by a similar method using a sample containing aknown concentration of an analyte.

[Method B]:

A known concentration of a detectable substance as an internal standardis added to a solution containing a sample having an analyte and notless than one kind of CFSs or a solution containing a labeled analoguein the above-described step (1) of [method A], and the above-describedsteps (1) to (3) are carried out using that solution containing theinternal standard. The amount of a labeling substance contained in theseparated complex B or the amount of a labeling substance contained in alabeled analogue not involved in formation of said complex B is measuredby a method corresponding to property (kind) of a labeling substance,and the amount of an analyte in a sample is calculated by comparing themeasurement result (measurement value) with the amount of said substanceadded as internal standard.

(m) A Case when a Labeled Analogue and a Reaction Improvement CFS areUsed.

In this case, for example, it may be carried out as the following[method A] or [method B].

[Method A]:

(1) (a) A solution containing a sample having an analyte, and not lessthan one kind of reaction improvement CFSs, and (b) a solutioncontaining a labeled analogue are introduced and arranged into acapillary, as the above-described step (1) in the case (m) of “1-6.Specific methods for forming a complex”,

(2) Subsequently, said labeled analogue is electrophoretically contactedwith said reaction improvement CFS (namely, said labeled analogue iselectrophoretically contacted with said reaction improvement CFS notinvolved in the formation of a complex (complex A) with said analyte inthe said solution including a sample having an analyte, and not lessthan one kind of reaction improvement CFS) to form the complex B betweensaid labeled analogue and reaction improvement CFS, as theabove-described step (2) in the case (m) of “1-6. Specific methods forforming a complex”,

(3) Said complex B and said labeled analogue not involved in formationof said complex are separated by further electric movement (migration),as the above-described step (3) in the case (m) of “2-2. specificmethods for separation”, and

(4) The amount of a labeling substance contained in the separatedcomplex B or the amount of a labeling substance contained in a labeledanalogue not involved in formation of said complex B is measured by amethod corresponding to property (kind) of a labeling substance, and theamount of an analyte in a sample is calculated by applying themeasurement result (measurement value) to a calibration curve showingrelation between the amount of an analyte and the amount of a labelingsubstance, obtained by a similar method using a sample containing aknown concentration of an analyte.

[Method B]:

A known concentration of a detectable substance as an internal standardis added to a solution containing a sample having an analyte and notless than one kind of reaction improvement CFSs or a solution containinga labeled analogue in the above-described step (1) of [method A], andthe above-described steps (1) to (3) are carried out using that solutioncontaining the internal standard. The amount of a labeling substancecontained in the separated complex B or the amount of a labelingsubstance contained in a labeled analogue not involved in formation ofsaid complex B is measured by a method corresponding to property (kind)of a labeling substance, and the amount of an analyte in a sample iscalculated by comparing the measurement result (measurement value) withthe amount of said substance added as internal standard.

(n) A Case when a Labeled Analogue, a CFS and a Reaction Improvement CFSare Used.

In this case, for example, it may be carried out as the following[method A] or [method B].

[Method A]:

(1) (a) A solution containing a sample having an analyte and not lessthan one kind of a CFS (or not less than one kind of a reactionimprovement CFS), (b) a solution containing a labeled analogue and (c) asolution containing not less than one kind of a reaction improvement CFS(or not less than one kind of a CFS) are introduced and arranged into acapillary, as the above-described step (1) in the case (n) of “1-6.Specific methods for forming a complex”,

(2) Subsequently, said analyte and labeled analogue areelectrophoretically contacted with said CFS and reaction improvement CFS[namely, a comple between said analyte and CFS (or reaction improvementCFS) in said solution (a) is electrophoretically contacted with saidreaction improvement CFS (or CFS) in said solution (c), and said labeledanalogue is electrophoretically contacted with said CFS (or reactionimprovement CFS) not involved in the formation of said complex with saidanalyte in said solution (a) and said reaction improvement CFS (or CFS)in said solution (c)], as the above-described step (2) in the case (n)of “1-6. Specific methods for forming a complex”,

(3) Said complex B and said labeled analogue not involved in formationof said complex B are separated by further electric movement(migration), as the above-described step (3) in the case (n) of “2-2.specific methods for separation”, and

(4) The amount of a labeling substance contained in the separatedcomplex B or the amount of a labeling substance contained in a labeledanalogue not involved in formation of said complex B is measured by amethod corresponding to property (kind) of a labeling substance, and theamount of an analyte in a sample is calculated by applying themeasurement result (measurement value) to a calibration curve showingrelation between the amount of an analyte and the amount of a labelingsubstance, obtained by a similar method using a sample containing aknown concentration of an analyte.

[Method B]:

A known concentration of a detectable substance as an internal standardis added to at least one selected from a solution containing a samplehaving an analyte and not less than one kind of a CFS (or not less thanone kind of a reaction improvement CFS), a solution containing a labeledanalogue and a solution containing not less than one kind of a reactionimprovement CFS (or not less than one kind of a CFS) in theabove-described step (1) of [method A], and the above-described steps(1) to (3) are carried out using that solution containing the internalstandard. The amount of a labeling substance contained in the separatedcomplex B or the amount of a labeling substance contained in a labeledanalogue not involved in formation of said complex B is measured by amethod corresponding to property (kind) of a labeling substance, and theamount of an analyte in a sample is calculated by comparing themeasurement result (measurement value) with the amount of said substanceadded as internal standard.

(o) A Case when a Reaction Improvement Analogue and a Labeled CFS areUsed.

In this case, for example, it may be carried out as the following[method A] or [method B].

[Method A]:

(1) (a) A solution containing a sample having an analyte, and not lessthan one kind of labeled CFSs, and (b) a solution containing a reactionimprovement analogue are introduced and arranged into a capillary, asthe above-described step (1) in the case (o) of “1-6. Specific methodsfor forming a complex”,

(2) Subsequently, said reaction improvement analogue iselectrophoretically contacted with labeled CFS (namely, said reactionimprovement analogue is electrophoretically with said labeled CFS notinvolved in the formation of a complex (complex A) with said analyte inthe said solution including a sample having an analyte, and not lessthan one kind of labeled CFSs) to form the complex B between saidreaction improvement analogue and labeled CFS, as the above-describedstep (2) in the case (o) of “1-6. Specific methods for forming acomplex”,

(3) Said complex B and said complex A are separated by further electricmovement (migration), as the above-described step (3) in the case (o) of“2-2. specific methods for separation”, and

(4) The amount of a labeling substance contained in the separatedcomplex B or the amount of a labeling substance contained in theseparated complex A is measured by a method corresponding to property(kind) of a labeling substance, and the amount of an analyte in a sampleis calculated by applying the measurement result (measurement value) toa calibration curve showing relation between the amount of an analyteand the amount of a labeling substance, obtained by a similar methodusing a sample containing a known concentration of an analyte.

[Method B]:

A known concentration of a detectable substance as an internal standardis added to a solution containing a sample having an analyte and notless than one kind of a labeled CFS or a solution containing a reactionimprovement analogue in the above-described step (1) of [method A], andthe above-described steps (1) to (3) are carried out using that solutioncontaining the internal standard. The amount of a labeling substancecontained in the separated complex B or the amount of a labelingsubstance contained in said the separated complex A is measured by amethod corresponding to property (kind) of a labeling substance, and theamount of an analyte in a sample is calculated by comparing themeasurement result (measurement value) with the amount of said substanceadded as internal standard.

A method for measurement of the present invention may be carried out inaccordance with the above-described known methods themselves except byusing a method for separation of the present invention, and reagents tobe used may also be selected, as appropriate, in accordance with knownmethods themselves.

4. A Kit of the Present Invention

A kit of the present invention is one to be used for carrying out theabove-described method for forming a complex, method for separation andmethod for measuring of the present invention.

As such a kit, the following items should be included:

(1) At least (i) the above-described CFS, (ii) if necessary an analogue,and (iii) an instruction book to be used in the above-described methodfor forming a complex, method for separation and method for measuring ofthe present invention; or

(2) (i) electrophoresis device (or microfluidic device) equipped with acapillary (channel) having at least a part enabling to carry out theabove-described step (1) of the present invention and a part enabling tocarry out the above-described step (2) of the present invention,preferably a capillary (channel) having a part enabling to carry out theabove-described step (1), a part enabling to carry out theabove-described step (2) and further a part enabling to carry out theabove-described step (3) of the present invention, (ii) a CFS, (iii) ifnecessary an analogue, and (iv) an instruction book to be used in theabove-described method for forming a complex, method for separation andmethod for measuring of the present invention.

In this connection, said “instruction book” means a handling manual(instruction manual) of said kit, attached documents (covering letter)or a pamphlet (leaflet), and the like, wherein features, principle,operation procedure, and the like, of a method of the present inventionare substantially described by writing or drawings, and the like.

Preferable embodiments and specific examples of these compositionelements are as described above.

Furthermore, a kit of the present invention may also contain reagentsother than the above. Such reagents include, for example, a buffersolution for electrophoresis separation, a reagent diluent, internalstandard, a calibrator (a standard solution), control, reagents (enzymesubstrate, coupling enzymes, and the like) for measurement of labelingsubstances (for example, enzyme, dyes, luminescent substances,fluorescent substances, and the like), reagents for focusing a detector,and the like, but not limited thereto.

The present invention is explained in more detail below by referring toExamples and Comparative Examples, however, the present invention shouldnot be limited thereby.

EXAMPLES Example 1 An Analyte (an Antigen)

α-fetoprotein (AFP) (manufactured by Wako Pure Chemical Industries,Ltd.)

[A Reaction Improvement CFS (a DNA Labeled Antibody)

According to the procedure shown in FIG. 1, anti-AFP antibody Fab′fragment bound with DNA was prepared.

Namely, a 250 bp DNA fragment introduced with an NH₂ group at the 5′terminal was purified first by a common method. Subsequently, the NH₂group introduced to the DNA fragment, and a succinimidyl group of asulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (Sulfo-SMPB) linker (alinker having a succinimidyl group and a maleimido group: manufacturedby Pierce Co.) were subjected to a reaction by a common method. Then, bygel filtration treatment, unreacted linkers were removed to give a 250bp DNA fragment bound with a linker. The resultant 250 bp DNA fragmentbound with a linker, and an anti-AFP antibody WA1 Fab′ fragment,prepared in advance using an anti-AFP antibody WA1 (manufactured by WakoPure Chemical Industries, Ltd.) in accordance with a common method, weresubjected to a reaction. The resultant reaction products were eachpurified using a DEAE column to prepare an anti-AFP antibody WA1 Fab′fragment bound with a 250 bp DNA fragment (a 250 bp DNA labeledantibody)

[A Labeled CFS (a Fluorescence Labeled Antibody)]

An anti-AFP antibody WA2 (manufactured by Wako Pure Chemical Industries,Ltd.) which recognizes an epitope of AFP different from a WA1 antibody,was treated by a common method to give an anti-AFP antibody WA2 Fab′fragment. A fluorescent substance Alexa647 (manufactured by MolecularProbes Inc.) was introduced to an amino group of said fragment by acommon method to prepare an Alexa647 labeled anti-AFP antibody WA2 Fab′fragment (a fluorescence labeled antibody).

[A Capillary Chip]

A capillary chip having a layout shown in FIG. 2 was produced accordingto a method described in “Technology and application of microchemistrychip”, T. Kitamori et al., published in 2004 (Maruzen Co., Ltd.) asfollows:

Namely, a photo resist film was formed on Si film which was formed on aquartz substrate. This photo resist was exposed using a mask having acapillary design (layout) shown in FIG. 2 and developed. Si at the part,where a photo resist was removed by development, was removed bysputtering, and then wet etching was carried out using a solution ofhydrogen fluoride to produce a capillary channel groove (capillary) atthe quartz substrate. After removing a photo resist and a Si filmremained on the quartz substrate, said quartz substrate and a coverplate having a hole for a fluid reservoir were adhered together by an HFbonding technique to produce a capillary chip.

In this connection, in FIG. 2, L1 and L2 show a well for introducing aleading buffer, S shows a well for introducing an electrophoresissample, R1 shows a well for introducing a reagent solution (a solutioncontaining a 250 bp DNA labeled antibody), and W1 and W2 show representa well for drain, respectively.

[Electrophoresis]

(1) A Electrophoresis Sample

Into a 0.5 mL tube, 1 μL of AFP having predetermined concentration, 1 μLof 2 μM a fluorescence labeled antibody and 8 μL of a leading buffercontaining 50 mM Cl⁻ ion were mixed to prepare 10 μL of a reactionsolution. The reaction solution was stood still on ice to be subjectedto an antigen-antibody reaction for about 30 minutes to form a[fluorescence labeled antibody-AFP] immune complex. In this connection,the final concentration of AFP was 0 pM, 25 pm, 50 pM or 100 pM, and thefinal concentration of the fluorescence labeled antibody was 200 nM.

The obtained reaction solution containing the immune complex was used asan electrophoresis sample.

(2) A Reagent Solution (a Solution Containing a 250 bp DNA LabeledAntibody)

A trailing buffer (containing 75 mM of HEPES) containing 20 nM of a 250bp DNA labeled antibody was used as a reagent solution.

(3) Procedure of Electrophoresis

a) Introduction of an Electrophoresis Sample and a Reagent Solution

Into an S well (a well for introducing an electrophoresis sample) shownin FIG. 1, 10 μL of an electrophoresis sample (a solution containing a[fluorescence labeled antibody-AFP] immune complex) was delivered bydrops, 10 μL of a reagent solution (a solution containing a DNA labeledantibody) was delivered by drops into an R1 well (a well for introducinga reagent solution), and 10 μL of a leading buffer was delivered bydrops into an L1 and L2 wells, respectively, and by application of apressure of −5 psi for 100 seconds between W1 (a well for drain) and W2(a well for drain) an electrophoresis sample, a reagent solution and aleading buffer were introduced into the channel. Arrangement relation ofan electrophoresis sample and a reagent solution in a capillary wasschematically shown in FIG. 3. In this connection, in FIG. 3, a shadedarea shows an arrangement area of an electrophoresis sample and a dottedarea shows an arrangement area of a reagent solution, respectively.

b) Concentration and Reaction

By applying a voltage of 312 V, 625 V or 2500 V between an R1 well andan L1 well, a 250 bp DNA labeled antibody in the reagent solutioncontacted with a [fluorescence labeled antibody-AFP] immune complex inthe electrophoresis sample, while concentrating a 250 bp DNA labeledantibody in a reagent solution, to form a [fluorescence labeledantibody-AFP-250 bp DNA labeled antibody] immune complex was formedwhile making concentrated a 250 bp DNA labeled antibody in a reagentsolution at 10° C.

In this connection, reaction time was about 200 seconds in applicationof a voltage of 312 V, about 100 seconds in application of a voltage of625 V, and about 25 seconds in application of a voltage of 2500 V.

c) Separation and Detection

When a [fluorescence labeled antibody-AFP-250 bp DNA labeled antibody]immune complex overtook through a crossing part of an L2 channel and amain channel, 2800 V was applied onto the L2 well and 300 V was appliedonto an L1 well, for 100 seconds to separate and detect said immunecomplex.

In this connection, the detection was carried out by serial measurementof fluorescent intensity by laser excitation of 635 nm at a capillarypart at 2 cm from the crossing part of the L2 channel, using a,fluorescent microscope (BX-50, manufactured from KS Olympus Co., Ltd.)

[Results]

FIG. 4 shows relation (linearity) between AFP concentration and peakarea, and FIG. 5 shows each of electrophoresis chromatogram when anelectrophoresis sample of an AFP concentration of 0 pM and 100 pM wasused. In this connection, in FIG. 4, vertical axis shows peak area andhorizontal axis shows AFP concentration, respectively. In addition, inFIG. 5, a solid line (-) shows the case when an electrophoresis sampleof an AFP concentration of 100 pM was used and a dotted line (• • •)shows the case when an electrophoresis sample of an AFP concentration of0 pM was used, respectively.

From FIGS. 4 and 5, it is found that a peak of a [fluorescence labeledantibody-AFP-250 bp DNA labeled antibody] immune complex is observed andpeak area thereof is proportional to AFP concentration. Namely, it canbe understood that an immune complex is electrophoretically formed in acapillary (channel) and there is no necessity to react and form theimmune complex in advance outside a capillary (channel).

In addition, Table 2 shows the relation between an applied voltage andan AFP reaction rate in concentration and reaction. As ComparativeExample, the AFP reaction rate obtained when these solutions are reactedfor 120 seconds by a conventional method introducing a plurality ofsolutions in a mixing capillary (channel) simultaneously to be subjectedto mixing and reaction (JP-A-2005-31070, and the like), was also shown.

In this connection, AFP reaction rate is a relative value obtained whenan AFP signal (peak area) was detected similarly as above after reactinga fluorescence labeled antibody, AFP and a 250 bp DNA labeled antibodyat 10° C. for 30 minutes in advance outside a capillary to obtain 10 μLof a reaction solution containing 200 nM of a fluorescence labeledantibody, 100 pM of AFP and 20 nM of a 250 bp DNA labeled antibody, andintroducing the obtained reaction solution from an S well, was taken as100%.

TABLE 2 Comparative Example Reaction (Conventional outside a Example(This invention) Method) capillary Impressed 2500 V 625 V 312 V — —Voltage Reaction About About About 120 secs. 30 mins. Time 25 secs. 100secs. 200 secs. AFP 145 346 435 — 460 Signal Reaction 32% 75% 95% 26%100% Rate

As is clear from Table 2, it is found that even in application of 2500 V(a reaction time of about 25 seconds), reaction rate equivalent to orover that in a conventional method can be obtained, and in the case ofapplication of 625 V (a reaction time of about 100 seconds) showingnearly the same reaction time in a conventional method, significantlyhigher reaction rate than that in a conventional method can be obtained.Namely, it can be understood that by a method of the present invention acomplex is formed in excellently higher reaction rate than in aconventional method.

Example 2 An Analyte (an Antigen)

The same one as in Example 1 was used.

[A Reaction Improvement CFS (a DNA Labeled Antibody]

The same one as in Example 1 was used.

[A labeled CFS (a fluorescence labeled antibody)]

The same one as in Example 1 was used.

[A Capillary Chip]

A capillary chip having a layout shown in FIG. 6 was produced accordingto a method described in “Technology and application of microchemistrychip”, T. Kitamori et al., published in 2004 (Maruzen Co., Ltd.) asfollows:

Namely, a photo resist film was formed on Si film which was formed on aquartz substrate. This photo resist was exposed using a mask having acapillary design (layout) shown in FIG. 6 and developed. Si at the part,where a photo resist was removed by development, was removed bysputtering, and then wet etching was carried out using a solution ofhydrogen fluoride to prepare a capillary channel groove (capillary) atthe quartz substrate. After removing a photo resist and a Si filmremained on the quartz substrate, said quartz substrate and a coverplate having a hole for a fluid reservoir were adhered together by an HFbonding technique to prepare a capillary chip.

In this connection, in FIG. 6, L1 and L2 show a well for introducing aleading buffer, and SR shows a well for introducing an electrophoresissample, the 1^(st) reagent solution (a solution containing a 250 bp DNAlabeled antibody) and 2^(nd) reagent solution (a solution containing afluorescence labeled antibody), respectively.

[Electrophoresis]

(1) A Electrophoresis Sample

Leading buffers (containing 50 mM of Cl⁻ ion) each containing 0 nM, 0.8nM, 4 nM, 20 nM, 50 nM and 100 nM of AFP were used as electrophoresissamples.

(2) The 1^(st) Reagent Solution (a Solution Containing a 250 bp DNALabeling Antibody)

A leading buffer (containing 50 mM of Cl⁻ ion) containing 100 nM of a250 bp DNA labeled antibody was used as the 1st reagent solution.

(3) The 2^(nd) Reagent Solution (a Solution Containing a FluorescenceLabeled Antibody)

A leading buffer (containing 50 mM of Cl⁻ ion) containing 400 nM of 250bp of a fluorescence labeled antibody was used as the 2^(nd) reagentsolution

(4) Procedure of Electrophoresis

a) Introduction of an Electrophoresis Sample, the 1^(st) ReagentSolution and the 2^(nd) Reagent Solution

After whole of the channel shown in FIG. 6 was filled with a leadingbuffer, 10 μL of the 2^(nd) reagent solution (a solution containing afluorescence labeled antibody) was delivered by drops into an SR well,and by application of a pressure of −5 psi for 2 seconds onto an L1 wellto introduce the 2^(nd) reagent solution were introduced into thechannel. Then, the 2^(nd) reagent solution in the SR well was replacedby 10 μL of the electrophoresis sample (a solution containing AFP), andsimilarly by application of a pressure of −5 psi for 2 seconds to the L1well the electrophoresis sample was introduced into the channel.Furthermore, the electrophoresis sample in the SR well was replaced by10 μL of the 1^(st) reagent solution (a solution containing a 250 bp DMAlabeled antibody), and by application of a pressure of −5 psi for 2seconds to the L1 well the 1^(st) reagent solution was introduced intothe channel. By this procedure, the 2^(nd) reagent solution zone, anelectrophoresis sample zone and the 1^(st) reagent solution zone wereformed in the channel in the order from the downstream side. Arrangementrelation of the electrophoresis sample, the 1^(st) reagent solution andthe 2^(nd) reagent solution in a capillary was schematically shown inFIG. 7. In this connection, in FIG. 7, a vertical line area shows anarrangement area of the 1^(st) reagent solution, a shaded area shows theelectrophoresis sample and a dotted area shows, the 2^(nd) reagentsolution, respectively.

Subsequently, the 1^(st) reagent solution in the SR well was replaced by10 μL of a trailing buffer containing 75 mM of HEPES, and by applicationof a pressure of −5 psi for 2 seconds onto the L1 well the trailingbuffer was introduced and arranged at the upstream side of the 1^(st)reagent solution zone.

b) Concentration and Reaction

By applying a voltage of 312 V between the SR well and the L1 well (awell for introducing a leading buffer), a DNA labeled antibody in the1^(st) reagent solution, AFP in the electrophoresis sample and afluorescence labeled antibody in the 2^(nd) reagent solution werecontacted, while concentrating these, to form a [fluorescence labeledantibody-AFP-250 bp DNA labeled antibody] immune complex at 10° C.

In this connection, reaction time is about 200 seconds.

c) Separation and Detection

When a [fluorescence labeled antibody-AFP-250 bp DNA labeled antibody]immune complex overtook through a crossing part of an L2 channel and amain channel, 2800 V was applied onto the L2 well and 300 V was appliedonto an L1 well, for 100 seconds to separate and detect said immunecomplex.

In this connection, the detection was carried out by serial measurementof fluorescent intensity by laser excitation of 635 nm at a capillarypart at 2 cm from the crossing part of the L2 channel, using afluorescent microscope (BX-50, manufactured from KS Olympus Co., Ltd.)

[Results]

FIG. 8 shows relation (linearity) between AFP concentration and peakarea, and FIG. 9 also shows relation (linearity) between AFPconcentration and peak area, in the low AFP concentration region(results for using electrophoresis samples of an AFP concentration of 0nM, 0.8 nM, 4 nM and 20 nM). In this connection, in FIGS. 8 and 9,vertical axis shows peak area and horizontal axis shows AFPconcentration, respectively. In addition, as Comparative Example, theresult obtained by similar detection as above after introducing areaction solution obtained by reaction of a fluorescence labeledantibody, AFP and a 250 bp DNA labeled antibody at 10° C. for 30 minutesin advance outside the capillary from the SR well to the capillary, wasalso shown in FIGS. 8 and 9. In this connection, the final concentrationof a fluorescence labeled antibody in the reaction was 200 nM, the finalconcentration of AFP was 0 nM. 0.8 nM, 4 nM, 20 nM, 50 nM or 100 nM, andthe final concentration of a 250 bp DNA labeled antibody was 20 nM.

In FIGS. 8 and 9, • mark shows the result obtained by a method inExample 2 and ∘ mark shows the result obtained by a method inComparative Example, respectively. In addition, in FIG. 9, a solid lineshows a regression line in the result obtained by method in Example 2and a dotted line shows a regression lines in the result obtained by amethod in Comparative Example, respectively.

From FIGS. 8 and 9, it is found that a peak of a [fluorescence labeledantibody-AFP-250 bp DNA labeled antibody] immune complex is observed andpeak area thereof is proportional to AFP concentration. Particularly, inthe case when an immune complex is formed in advance, and thenelectrophoresis treatment is carried out, the linearity is poor in thelow AFP concentration region (the detection values lie beneath theregression line), whereas good linearity is found to be obtained in anyof low and high concentration regions of AFP, in the case of the presentinvention, wherein an immune reaction is carried out in a capillaryelectrophoretically, and then electrophoresis treatment is carried out.

INDUSTRIAL APPLICABILITY

The present invention relates to a method for forming a complex betweenan analyte or an analogue thereof in a sample, and a substance formablea complex with said analyte or analogue thereof (a CFS), a method forseparating a complex formed, and a CFS or an analogue not involved information of said complex, along with a method for measuring an analytein a sample, based on the amount of a complex separated, or the amountof a CFS or an analogue not involved in formation of a complex.

In accordance with a method of the present invention, a reaction betweenan analyte or an analogue thereof in a solution, and a substanceformable a complex with said analyte or analogue thereof (a CFS) in asolution, can be carried out in a short time and in high reactionefficiency. As a result, separation of a complex with a substanceformable a complex with said analyte or analogue thereof (a CFS), and aCFS or an analogue not involved in formation of a complex becomespossible rapidly, simply and in high accuracy, and furthermore, highsensitivity measurement of an analyte in a sample becomes possible,based on the amount of separated complex or the amount of a CFS oranalogue not involved in formation of a complex.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a preparation scheme of a DNA labeled antibody [an anti-AFPantibody WA1 Fab′ fragment bound with a 250 bp DNA fragment (a reactionimprovement CFS)], prepared in Example 1.

FIG. 2 shows a layout of a capillary chip prepared in Example 1.

FIG. 3 shows an arrangement relation of an electrophoresis sample and areagent solution introduced in a capillary in Example 1.

FIG. 4 shows a relation (linearity) between AFP concentration and peakarea, obtained in Example 1.

FIG. 5 shows electrophoresis chromatograms in the cases of usingelectrophoresis samples with AFP concentrations of 0 pM and 100 pM,obtained in Example 1.

FIG. 6 shows a layout of a capillary chip prepared in Example 2.

FIG. 7 shows an arrangement relation of an electrophoresis sample, the1^(st) test sample and the 2^(nd) reagent solution introduced in acapillary in Example 2.

FIG. 8 shows a relation (linearity) between AFP concentration and peakarea, obtained in Example 2.

FIG. 9 shows a relation (linearity) between AFP concentration and peakarea in low AFP concentration regions (results for using electrophoresissamples of an AFP concentration of 0 nM, 0.8 nM, 4 nM and 20 nM),obtained in Example 2.

The invention claimed is:
 1. A method for separating a complexcomprising the following steps: (1) a step of arranging (a) a solutioncontaining an analyte or an analogue thereof and (b) a solutioncontaining not less than one kind of a substance capable of forming thecomplex with said analyte or said analogue thereof (the complex formingsubstance), each of (a) and (b) in a separate zone in a capillary, sothat by applying a voltage to said capillary the complex between saidanalyte or said analogue thereof and the complex forming substance areformed without mixing these solutions in advance; (2) a step of formingthe complex by electrophoretically concentrating said analyte or saidanalogue thereof and/or the complex forming substance by utilizing adifference in their electrophoretic mobility wherein said analyte orsaid analogue thereof and/or said complex forming substance are gatheredon application of a voltage onto a capillary by applying a voltage tosaid capillary, before these solutions are uniformly mixed to form thecomplex between said analyte or said analogue thereof and the complexforming substance; and (3) a step of separating said complex, and thecomplex forming substance not involved in the formation of said complexor the analogue not involved in the formation of said complex by furtherelectrical movement by using a different principle of electrophoresisfrom that in step (2), wherein step (2) is carried out by ITP(isotachophoresis) or FASS (Field Amplification Sample Stacking).
 2. Themethod according to claim 1, wherein at least said analyte after theapplied voltage is concentrated by not less than 1.5 times said analytebefore the applied voltage as a result of the application of voltage instep (2), said analogue after the applied voltage is concentrated by notless than 1.5 times said analogue before the applied voltage as a resultof the application of voltage in step (2), or said at least one kind ofthe complex forming substances after the applied voltage is concentratedby not less than 1.5 times said complex forming substance before theapplied voltage as a result of the application of voltage in step (2).3. The method according to claim 1, wherein not less than one kind ofthe complex forming substance is one bound with a labeling substanceand/or a reaction improvement substance capable of changingelectrophoretic mobility of said analyte or said analogue thereof. 4.The method according to claim 1, wherein not less than two kinds ofcomplex forming substances are used, and not less than two kinds ofsolutions containing each of such complex forming substances are used,and (1) at least one kind of the complex forming substances is one boundwith a labeling substance, and at least one kind of the other complexforming substance is one bound with a reaction improvement substancecapable of changing electrophoretic mobility of said analyte or saidanalogue thereof, or (2) at least one kind of the complex formingsubstance is one bound with a labeling substance and a reactionimprovement substance capable of changing electrophoretic mobility ofsaid analyte or said analogue thereof.
 5. The method according to claim1, wherein one kind of the complex forming substances is an antibody tosaid analyte or said analogue thereof, or protein binding to saidanalyte or said analogue thereof.
 6. The method according to claim 1,wherein in step (1), a solution containing a substance with a higherelectrophoretic mobility is arranged upstream of a solution containing asubstance with lower electrophoretic mobility.
 7. The method accordingto claim 1, wherein step (3) is carried out at a separation regionindependent of a part where step (2) is carried out in said capillary.8. The method according to claim 1, wherein step (3) is carried out bycapillary gel electrophoresis method.
 9. The method according to claim1, wherein said analogue is one bound with a labeling substance or areaction improvement substance capable of changing electrophoreticmobility of said analyte or said analogue thereof.
 10. The methodaccording to claim 1, wherein step (1) is carried out by applying avoltage onto the capillary, increasing and/or reducing pressure in thecapillary and/or relying on capillary phenomenon.
 11. A method forseparating a complex comprising the following steps: (1) a step ofarranging (a) a solution containing an analyte or an analogue thereofand (b) a solution containing not less than one kind of a substancecapable of forming the complex with said analyte or said analoguethereof (the complex forming substance), each of (a) and (b) in aseparate zone in a capillary, so that by applying a voltage to saidcapillary the complex between said analyte or said analogue thereof andthe complex forming substance are formed without mixing these solutionsin advance; (2) a step of forming the complex by electrophoreticallyconcentrating said analyte or said analogue thereof and/or the complexforming substance by utilizing a difference in their electrophoreticmobility wherein said analyte or said analogue thereof and/or saidcomplex forming substance are gathered on application of a voltage ontoa capillary by applying a voltage to said capillary, before thesesolutions are uniformly mixed to form the complex between said analyteor said analogue thereof and the complex forming substance; (3) a stepof separating said complex, and the complex forming substance notinvolved in the formation of said complex or the analogue not involvedin the formation of said complex by further electrical movement by usinga different principle of electrophoresis from that in step (2); and (4)a step of measuring the amount of thus separated complex, and/or theamount of the complex forming substance and/or the analogue not involvedin the formation of said complex to determine the amount of said analytebased on the result, wherein step (2) is carried out by ITP(isotachophoresis) or FASS (Field Amplification Sample Stacking). 12.The method according to claim 11, wherein at least said analyte afterthe applied voltage is concentrated by not less than 1.5 times saidanalyte before the applied voltage as a result of the application ofvoltage in step (2), said analogue after the applied voltage isconcentrated by not less than 1.5 times said analogue before the appliedvoltage as a result of the application of voltage in step (2), or saidat least one kind of the complex forming substances after the appliedvoltage is concentrated by not less than 1.5 times said complex formingsubstance before the applied voltage as a result of the application ofvoltage in step (2).
 13. The method according to claim 11, wherein notless than one kind of the complex forming substance is one bound with alabeling substance and/or a reaction improvement substance capable ofchanging electrophoretic mobility of said analyte or said analoguethereof.
 14. The method according to claim 11, wherein not less than twokinds of complex forming substances are used, and not less than twokinds of solutions containing each of such complex forming substancesare used, and (1) at least one kind of the complex forming substances isone bound with a labeling substance, and at least one kind of the othercomplex forming substance is one bound with a reaction improvementsubstance capable of changing electrophoretic mobility of said analyteor said analogue thereof, or (2) at least one kind of the complexforming substance is one bound with a labeling substance and a reactionimprovement substance capable of changing electrophoretic mobility ofsaid analyte or said analogue thereof.
 15. The method according to claim11, wherein one kind of the complex forming substances is an antibody tosaid analyte or said analogue thereof, or protein binding to saidanalyte or said analogue thereof.
 16. The method according to claim 11,wherein in step (1), among said solutions, a solution containing asubstance with a higher electrophoretic mobility is arranged upstream ofa solution containing a substance with lower electrophoretic mobility.17. The method according to claim 11, wherein step (3) is carried out ata separation region independent of a part where step (2) is carried outin said capillary.
 18. The method according to claim 11, wherein step(3) is carried out by capillary gel electrophoresis method.
 19. Themethod according to claim 11, wherein said analogue is one bound with alabeling substance or a reaction improvement substance capable ofchanging electrophoretic mobility of said analyte or said analoguethereof.